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The increased expression of integrin ?6 (ITGA6) enhances drug resistance in EVI1(high) leukemia.

ABSTRACT: Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1(high)) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin ?6 (ITGA6) was upregulated over 10-fold in EVI1(high) leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1(high) leukemia cells. We found that the adhesion ability of EVI1(high) leukemia cells to laminin increased with the increased expression of ITGA6 and integrin ?4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1(high) leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1(low) leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1(high) AML cells downregulated their cell adhesion ability; however, the EVI1(high) AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1(high) leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1(high) AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1(high) AML.

SUBMITTER: Yamakawa N

PROVIDER: S-EPMC3266272 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The increased expression of integrin α6 (ITGA6) enhances drug resistance in EVI1(high) leukemia.

Yamakawa Norio N Kaneda Kazuko K Saito Yusuke Y Ichihara Emi E Morishita Kazuhiro K

PloS one 20120125 1

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1(high)) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1(high) leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhe ...[more]

PMID: 22295105

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Project description:Central nervous system leukemia (CNS-L) is caused by leukemic cells infiltrating into the meninges or brain parenchyma and remains the main reason for disease relapse. Currently, it is hard to detect CNS-L accurately by clinically available imaging models due to the relatively low amount of tumor cells, confined blood supply, and the inferior glucose metabolism intensity. Recently, integrin α6-laminin interactions have been identified to mediate CNS-L, which suggests that integrin α6 may be a promising molecular imaging target for the detection of CNS-L. The acute lymphoblastic leukemia (ALL) cell line NALM6 stabled and transfected with luciferase was used to establish the CNS-L mouse model. CNS-L-bearing mice were monitored and confirmed by bioluminescence imaging. Three of our previously developed integrin α6-targeted peptide-based molecular imaging agents, Cy5-S5 for near-infrared fluorescence (NIRF), Gd-S5 for magnetic resonance (MR), and 18F-S5 for positron emission tomography (PET) imaging, were employed for the molecular imaging of these CNS-L-bearing mice. Bioluminescence imaging showed a local intensive signal in the heads among CNS-L-bearing mice; meanwhile, Cy5-S5/NIRF imaging produced intensive fluorescence intensity in the same head regions. Moreover, Gd-S5/MR imaging generated superior MR signal enhancement at the site of meninges, which were located between the skull bone and brain parenchyma. Comparatively, MR imaging with the clinically available MR enhancer Gd-DTPA did not produce the distinguishable MR signal in the same head regions. Additionally, 18F-S5/PET imaging also generated focal radio-concentration at the same head regions, which generated nearly 5-times tumor-to-background ratio compared to the clinically available PET radiotracer 18F-FDG. Finally, pathological examination identified layer-displayed leukemic cells in the superficial part of the brain parenchyma tissue, and immunohistochemical staining confirmed the overexpression of the integrin α6 within the lesion. These findings suggest the potential application of these integrin α6-targeted molecular imaging agents for the accurate detection of CNS-L.

Project description:Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.

Dataset Information

The increased expression of integrin ?6 (ITGA6) enhances drug resistance in EVI1(high) leukemia.

Publications

The increased expression of integrin α6 (ITGA6) enhances drug resistance in EVI1(high) leukemia.

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