The Expression And Function Of Human Endogenous Retrovirus HERV-K Np9Protein In Human Leukemias

Leukemia is a common hematopoietic tumor of unknown etiology, which is estimated to affect250,000people worldwide. Unfortunately, the majority of leukemia patients will die due to drug resistance or relapse. Currently, the uncertain etiology and molecular mechanisms greatly hamper development of efficient drugs and regimens for curing this malignant disease. Among all the studies on etiology, like virus, ionizing radiation, chemicals and genetic factors, viral etiology has been long considered as the most significant and attractive topic. It has been demonstrated that the retroviruses have been etiologically linked to a number of animal leukemias, and a rare type of human leukemia:human adult T cell leukemia/lymphoma. However, whether retroviruses have been etiologically linked to human common leukemia such as acute and chronic myeloid leukemia, acute and chronic lymphoblastic leukemia remain elusive.Although the majority of HERVs (human endogenous retrovirus sequences, HERVs) are dysfunctional due to the accumulation of multiple nonsense mutations, some are still active and may have a potential function, notably the human endogenous retrovirus type K (HERV-K) family. The viral Np9transcript, a small regulatory gene encoded by HERV-K type I, has been shown to be exclusively present in tumors and transformed cells. Our previous studies have demonstrated the presence of retrovirus particles in primary leukemia cells from patients with acute myeloid leukemia (AML). In addition, the viral Tax protein of human T-Cell leukemia virus type1(HTLV-1) has been implicated in leukemogenesis. However, Np9protein expression and its possible role in human common leukemia have not been studied. The aim of this study was to investigate whether the Np9protein is expressed in human leukemia cells and whether the viral Np9plays a role in cell growth of human common leukemia including myeloid and lymphoblastic leukemia. If so, we then further assess possible molecular mechanism by which Np9promotes the growth of leukemia cells. The results of this study may provide a potential novel therapeutic target and opens new perspectives to unravel the etiology of human leukemia.Part Ⅰ The expression of Np9protein in human leukemiasWe examined the expression of Np9in14various human hematopoietic malignant cell lines,16different solid tumor cell lines, primary leukemia cells from a cohort of50patients with various leukemia, and normal hematopoietic cell samples, including CD34+hematopoietic stem cells (HSCs), from22healthy adult donors.78.57%hematopoietic tumor cell lines showed high levels of Np9protein, including3chronic myeloid leukemia (CML)(K562, K562/adr, KCL-22M),3acute myeloid leukemia (AML)(HL-60, NB4, Kasumi-1),3acute lymphoblastic leukemia (ALL)(Jurkat, Molt-4,119), and2multiple myeloma (MM)(8226, KM3).18.75%tumor cell lines:U87(glioblastoma), A549(lung cancer), and PANC-1(pancreatic adenocarcinoma) also showed high levels of Np9protein. Significantly,56%primary leukemia samples showed expression of Np9protein. In contrast, Np9protein was rarely detected in normal hematopoietic cells from healthy individuals. These results indicate that Np9protein expression is universal in both human leukemia cell lines and primary leukemia samples, but rarely in normal blood cells. We next examined the Np9protein levels in CD34+leukemia stem/progenitor cells in primary leukemia samples. Np9protein levels were positively correlated with CD34+leukemia stem/progenitor cells (P=0.003).We next asked whether Np9-positive leukemia samples also express pol-env polyprotein, env and transmembrane proteins using a specific antibody against a defined domain of Env, the transmembrane (TM) domain. Leukemia cell lines also expressed leukemia-specific pol-env polyprotein, env protein and transmembrane proteins (TM). In contrast, these leukemia-specific viral proteins were not detected in normal blood cells from12normal individuals.We then used real-time PCR to test whether Np9transcription could be up-regulated in K562by effect of different factors such as hormone, alcohol, demethylating substance and retroviral inducers. Dexamethasone,alcohol and IdUR up-regulated the expression of Np9as the concentration increased.5-Aza did not affect the transcription levels.Part ⅡNp9is essential for survival and growth of leukemia cells in vitro and in vivoTo determine whether Np9is essential for the survival of leukemia cells, we suppressed Np9expression by using shRNAs against the viral Np9gene. Recombinant lentiviruses transcribing short hairpin RNAs against Np9were generated and transduced to Np9protein-expressing human myeloid leukemia K562and lymphoblastic leukemia Jurkat cells. Proliferation of leukemia cells were measured by MTS assay. The results showed a marked growth inhibition of both K562and Jurkat cells by Np9-specific shRNAs.To assess whether Np9promotes the growth of leukemia cells, we generated recombinant lentiviruses expressing Np9and transduced to Np9-negative human leukemia Raji cells and determined growth curves of Raji cells with over-expression of Np9and found that Np9significantly promoted the growth of Raji cells.We next examined the effects of Np9expression on the colony-forming ability of tumor cells using colony forming assay. In contrast to control, Np9over-expression induced an increase of in the number and size of colonies.We also evaluated the subcellular localization of the Np9protein, and found that viral Np9protein was predominantly localized in the nucleus of leukemia cells, but a small amount of viral Np9protein was also observed in the cytoplasm of tumor cells.To confirm whether Np9promotes the growth of leukemia cells in vivo, Raji cells with Np9over-expression or control cells were injected subcutaneously into immunocompromized NOD-SCID mice following standard protocols. We observed that tumors in mice (n=6) that received cells with Np9grew much faster and tumor weight was1.74g±0.42g within25days. In contrast, tumors in mice that received empty vector as negative control grew slowly and tumor weight was0.62g±0.18g within25days. These data clearly indicate that Np9expression strongly supports and promotes the growth of leukemia cells in NOD-SCID mice.Part Ⅲ The molecular mechanism by which Np9promotes the growth of leukemia cellsWe examined whether Np9expression co-activates Wnt/β-catenin, Notch1, ras/ERK and c-myc/AKT signaling pathways, which are aberrantly activated in human leukemia, in4different leukemia cell lines (K562, Kasumi-1, NB4and Jurkat) with high levels of Np9expression. The results showed that these leukemia cell lines had a co-activation of multiple leukemia-associated signaling pathways. In contrast to leukemia cells, no obvious activation of these leukemia-related signaling pathways was observed in normal blood cells from healthy donors. To address whether Np9affects these signaling components, we transfected Np9-negative Raji cells using Np9expression plasmid and observed that over-expression of Np9led to a marked up-regulation of β-catenin, phospho-ERK (pERK), c-myc and cleaved Notch1with a concomitant decrease of Numb. To validate these results, we then silenced Np9gene expression in K562and Jurkat leukemia cells using shRNAs. The results revealed that Np9silencing caused significant decreases of expression levels of c-myc, pERKl, phospho-Akt (pAkt) and cleaved Notchl with a concomitant increase of Numb.To characterize Np9protein sequences, we found that there were five intact ORFs for Np9protein. Alignment analysis showed that amino acid sequences were highly similar. We identified a Grb2SH3(N)-typical motif (PxxPxR) among these deduced Np9proteins which was absolutely conserved throughout all Np9proteins indicating a potential role in Np9-mediated leukemogenesis.