Functional investigation of ANKHD1 in malignant neoplasms

Author(s)

Patricia Cristina Rodrigues

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2010-02-26

Examining board members

Sara Teresinha Olalla Saad; José Andrés Yunes; Licio Augusto Velloso; Serge Benichou; Gisele Wally Braga Colleoni

• Advisor: Sara Teresinha Olalla Saad

Abstract

The human protein ANKHD1 (Ankyrin Repeat Single KH Domain containing 1) was initially described in Human prostate adenocarcinoma cells (LNCaP), and can also be expressed in many different leukemic cell lines. ANKHD1 is homologous to the Drosophila melanogaster Mask protein (Multiple Ankyrin repeat and single KH domain), related to differentiation, proliferation and survival in photoreceptors of Drosophila eyes. Direct interactions between ANKHD1 and SHP2, a protein that plays a critical role in hematopoiesis, have been described in the myeloid cell line K562, in prostate adenocarcinome, LNCaP, and in multiple myeloma cell line RPM1 8226. Nevertheless, little is known of the function of ANKDH1 and its role in leukemogenesis. The purpose of this work was to investigate the involvement of ANKHD1 in biological processes related to leukemogenesis. For this purpose, we used two main methodological approaches: ANKHD1 inhibition and overexpression. The inhibition of ANKHD1 in LNCaP cell lines was followed by the tracking through microarray of the remaining associated genetic alterations. Microarray revealed 706 genes with upregulated expression and 159 genes with downregulated expression; the genes were gathered in 26 functional groups and many genes, known for regulating cell cycle and apoptosis, had alterations in their expression profile. Importantly, inhibition of ANKHD1 resulted in significant increase in the expression of a large number of Histones, suggesting its role in epigenetic control. Among the validated modulations, some were also observed in the samples collected from leukemia patients. In this study, the downregulation of ANKHD1 was demonstrated in samples collected from 38 acute leukemia patients when compared to normal bone marrow cells. As this leukemic sample represented a natural model of inhibition of ANKHD1, the expression of other genes processed through array were tested, and the BMF gene, which was upregulated in the array, was also more elevated in leukemia samples. Simultaneously, cells of acute leukemia and myelodysplasia were subjected to the action of various drugs used to treat these diseases showing increased expression of ANKHD1. These data together with those observed in the microarray suggest the participation of ANKHD1 towards JAK/STAT1 and pathways of epigenetic control. The second approach used in this work was the ANKHD1overexpression, isoforms1 and 2, in tumor lines, followed by the detection of protein expression in cotransfection with Siva and Vpr genes. The proteins Siva and Vpr have been recently described as being capable of physical interaction with the isoforms 1 and 2 of ANKHD1, respectively. The role of inducing apoptosis in T lynphocytes was attributed to both Siva and Vpr. By cotrasfecting Siva and Vpr with ANKHD1 a reduction of ANKHD1 expression to isoform 1 as well as to isoform 2 (western blotting) was obtained and isoform 1 of ANKHD1 was observed to be citoplasmatic, such as occurs with drosophila Mask, although isoform 2 happened to be predominantly nuclear in the fluorescence microscope. Functional analysis of overexpression of ANKHD1 followed by FACS analysis suggests reduction of apoptosis in leukemic cells. Finally, this study identified many possible participants in the signaling pathway of ANKHD1 by microarray analysis, and related the reduction of its expression in leukemia patients, as well as the increasing of its expression in leukemic cell lines under the effect of many different treatments currently used against hematopoietic disorders. We also described the increasing of BMF expression in the same samples where we found a reduction of ANKHD1, corroborating all the data we obtained from the ANKHD1 inhibition microarray. In addition, we described, for the first time, the nuclear location of ANKHD1's isoform 2, confirming all the computer-aided predictions regarding the location of ANKHD1. The results herein described suggest that ANKHD1 could be involved with the abnormal phenotype of leukemic cells and these results could guide further studies towards elucidating the specific functions of ANKHD1 in hematopoiesis (AU)