Role of Misfolded - Nuclear receptor co-repressor (N-Cor) induced transcriptional de-regulation in the pathogenesis of acute monocytic leukemia (AML-M5)

Ph.DDOCTOR OF PHILOSOPH

Role of Chaperone Mediated Autophagy (CMA) in the Degradation of Misfolded N-CoR Protein in Non-Small Cell Lung Cancer (NSCLC) Cells

Nuclear receptor co-repressor (N-CoR) plays important role in transcriptional control mediated by several tumor suppressor proteins. Recently, we reported a role of misfolded-conformation dependent loss (MCDL) of N-CoR in the activation of oncogenic survival pathway in acute promyelocytic leukemia (APL). Since N-CoR plays important role in cellular homeostasis in various tissues, therefore, we hypothesized that an APL like MCDL of N-CoR might also be involved in other malignancy. Indeed, our initial screening of N-CoR status in various leukemia and solid tumor cells revealed an APL like MCDL of N-CoR in primary and secondary tumor cells derived from non-small cell lung cancer (NSCLC). The NSCLC cell specific N-CoR loss could be blocked by Kaletra, a clinical grade protease inhibitor and by genistein, an inhibitor of N-CoR misfolding previously characterized by us. The misfolded N-CoR presented in NSCLC cells was linked to the amplification of ER stress and was subjected to degradation by NSCLC cell specific aberrant protease activity. In NSCLC cells, misfolded N-CoR was found to be associated with Hsc70, a molecular chaperone involved in chaperone mediated autophagy (CMA). Genetic and chemical inhibition of Lamp2A, a rate limiting factor of CMA, significantly blocked the loss of N-CoR in NSCLC cells, suggesting a crucial role of CMA in N-CoR degradation. These findings identify an important role of CMA-induced degradation of misfolded N-CoR in the neutralization of ER stress and suggest a possible role of misfolded N-CoR protein in the activation of oncogenic survival pathway in NSCLC cells

Role of Misfolded N-CoR Mediated Transcriptional Deregulation of Flt3 in Acute Monocytic Leukemia (AML)-M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic multi-protein complex involved in transcriptional control. Flt3, a key regulator of hematopoietic cell growth, is frequently deregulated in AML (acute myeloid leukemia). Here, we report that loss of N-CoR-mediated transcriptional control of Flt3 due to misfolding, contributes to malignant growth in AML of the M5 subtype (AML-M5). An analysis of hematopoietic genes in AML cells led to the identification of Flt3 as a transcriptional target of N-CoR. Flt3 level was inversely related to N-CoR status in various leukemia cells. N-CoR was associated with the Flt3 promoter in-vivo, and a reporter driven by the Flt3 promoter was effectively repressed by N-CoR. Blocking N-CoR loss with Genistein; an inhibitor of N-CoR misfolding, significantly down-regulated Flt3 levels regardless of the Flt3 receptor mutational status and promoted the differentiation of AML-M5 cells. While stimulation of the Flt3 receptor with the Flt3 ligand triggered N-CoR loss, Flt3 antibody mediated blockade of Flt3 ligand-receptor binding led to N-CoR stabilization. Genetic ablation of N-CoR potentiated Flt3 ligand induced proliferation of BA/F3 cells. These findings suggest that N-CoR-induced repression of Flt3 might be crucial for limiting the contribution of the Flt3 signaling pathway on the growth potential of leukemic cells and its deregulation due to N-CoR loss in AML-M5, could contribute to malignant growth by conferring a proliferative advantage to the leukemic blasts. Therapeutic restoration of N-CoR function could thus be a useful approach in restricting the contribution of the Flt3 signaling pathway in AML-M5 pathogenesis

Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

10.1371/journal.pone.0070891PLoS ONE88-POLN

Biology of Cancer-Testis Antigens and Their Therapeutic Implications in Cancer

10.3390/cells12060926CELLS12

Misfolded N-CoR is linked to the ectopic reactivation of CD34/Flt3-based stem-cell phenotype in promyelocytic and monocytic acute myeloid leukemia

Nuclear receptor co-repressor (N-CoR) is the key component of generic co-repressor complex essential for the transcriptional control of genes involved in cellular hemostasis. We have recently reported that N-CoR actively represses Flt3, a key factor of hematopoietic stem cells (HSC) self-renewal and growth; and that de-repression of Flt3 by the misfolded N-CoR plays important role in the pathogenesis of promyelocytic and monocytic acute myeloid leukemia (AML). The leukemic cells derived from the promyelocytic and monocytic AML are distinctly characterized by the ectopic reactivation of stem cell phenotypes in relatively committed myeloid compartment. However, the molecular mechanism underlying this phenomenon is not known. Here, we report that N-CoR function is essential for the commitment of primitive hematopoietic cells to the cells of myeloid lineage, and that loss of N-CoR function due to misfolding is linked to the ectopic reactivation of generic stem cell phenotypes in promyelocytic and monocytic AML. Analysis of N-CoR and Flt3 transcripts in mouse hematopoietic cells revealed a positive correlation between N-CoR level and the commitment of myeloid cells and an inverse correlation between N-CoR and Flt3 levels in primitive as well as committed myeloid cells. Enforced N-CoR expression in mouse HSCs inhibited their growth and self-renewal potentials and promoted maturation towards cells of myeloid lineage, suggesting a role of N-CoR in the commitment of cells of myeloid lineage. In contrast to AML cells with natively folded N-CoR, primary and secondary promyelocytic and monocytic AML cells harboring the misfolded N-CoR were highly positive for Flt3 and myeloid antigen based HSC marker CD34. Genetic and therapeutic restoration of N-CoR conformation significantly down-regulated the CD34 levels in monocytic AML cells, suggesting an important role of N-CoR in the suppression of CD34 based hematopoietic stem cell phenotypes. These finding collectively suggest that N-CoR is crucial for the commitment of primitive hematopoietic cells to cells of myeloid lineage and that misfolded N-CoR may contribute to transformation of committed myeloid cells through the ectopic reactivation of Flt3/CD34 based stem cell phenotypes in promyelocytic and monocytic AML. Moreover, these finding provide novel mechanistic insights into the formation of leukemic stem cells in a subset

BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression

Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4), a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment

Restoration of N-CoR function attenuates the proliferative properties of AML-M5 cells regardless of the Flt3 receptor mutational status.

<p><i>A</i>, Genistein stabilized N-CoR in all 5 AML-M5 cell lines at 50 µM with a concurrent down-regulation of the Flt3 receptor expression as determined via western blotting. <i>B</i>, Restoration of N-CoR function in AML-M5 cells attenuated the proliferative potential of AML-M5 cells. Proliferative capacity of N-CoR positive AML cells HL-60 and N-CoR negative AML-M5 cells after treatment with Genistein at 72 hours was determined via growth proliferation assay (MTT). Growth inhibition was most pronounced at 50 µM in AML-M5 cells while at the same dose, growth inhibitory effects on N-CoR positive HL-60 was less pronounced. Results are representative of 3 independent experiments and asterisks represent p<0.05. <i>C</i>, Morphology of N-CoR positive HL-60 and N-CoR null AML-M5 cells (THP-1, Nomo-1, MV-4-11, SigM5, MM1) as determined via Wright-Giemsa staining after treatment with 50 µM Genistein for 72 hours.</p

N-CoR loss promotes growth potential, which is amplified by Flt3 signaling activation.

<p><i>A</i>, Levels of N-CoR and Flt3 protein in Ba/F3 cells transfected with control or N-CoR siRNA were determined via western blotting assay with an anti-Flt3 antibody that recognizes the murine form of the receptor and anti-N-CoR antibody. <i>B</i>, N-CoR loss mediated Flt3 expression enhanced the IL-3 independent growth potential of BA/F3 cells. Effect of IL-3 independent growth of Ba/F3 cells transfected with control or N-CoR siRNA (population from 4A) with and without Flt3 ligand stimulation were determined by cell proliferation assay. The Y-axis of the graph represents the proliferation index of viable cells, and the duration of culture is plotted on the X-axis. The symbols used in the graph are as follows: Control siRNA transfected cells treated with vehicle (<>\raster="rg1"<>), Control siRNA transfected cells stimulated with Flt3 ligand (<>\raster="rg2"<>), N-CoR siRNA transfected cells treated with vehicle (<>\raster="rg3"<>) and N-CoR siRNA transfected cells stimulated with Flt3 ligand (<>\raster="rg4"<>). The values presented in each graph are average of three independent experiments. <i>C</i>, Flt3 stimulation leads to N-CoR loss. Levels of N-CoR and Flt3 proteins in 293T cells treated with vehicle or Flt3 ligand (30 ng/ml) was determined by western blotting assay. <i>D</i>, Blocking Flt3 stimulation leads to N-CoR stabilization in THP-1 cells. Effect of Flt3 antibody on the levels of N-CoR and Flt3 proteins in THP-1 cells treated with vehicle or Flt3 ligand (30 ng/ml) was determined by western blotting assay.</p

Restoration of N-CoR function in THP-1 cells, down-regulated Flt3 levels and induced differentiation.

<p><i>A</i>, Flt3 levels were down-regulated at both the protein and mRNA levels after Genistein treatment. Level of Flt3 expression was determined via western blotting and RT-PCR analysis. <i>B</i>, Genistein inhibited the proliferation of THP-1 cells in a dose dependent manner when determined via growth proliferation assay (MTT). Results are representative of 3 independent experiments and asterisks represent p<0.05. <i>C</i>, Nuclear morphology of THP-1 cells treated with Genistein for the duration of 72 hours in a dose dependent manner was determined in Wright–Giemsa assay. The arrowheads mark the indented-shaped nucleus of differentiated cells. <i>D</i> & <i>E</i>, CD14 levels in THP-1 cells treated with Genistein for 72 hours in a dose dependent manner was determined by FACS (<i>D</i>) and RT-PCR (<i>E</i>) analysis. <i>F</i>, N-CoR, Flt3 and CD14 levels in THP-1 cells transfected with N-CoR siRNA and treated with 50 µM Genistein for 72 hours was determined by western blotting assay and RT-PCR analysis.</p