Effects of notch signaling on regulation of myeloid cell differentiation in cancer

Abstract

Functionally altered myeloid cells play an important role in immune suppression in cancer, in angiogenesis, and in tumor cells' invasion and metastases. Here, we report that inhibition of Notch signaling in hematopoietic progenitor cells (HPC), myeloid-derived suppressor cells (MDSC), and dendritic cells is directly involved in abnormal myeloid cell differentiation in cancer. Inhibition of Notch signaling was caused by the disruption of the interaction between Notch receptor and transcriptional repressor CSL, which is normally required for efficient transcription of target genes. This disruption was the result of serine phosphorylation of Notch. We demonstrated that increased activity of casein kinase 2 (CK2) observed in HPC and in MDSC could be responsible for the phosphorylation of Notch and downregulation of Notch signaling. Inhibition of CK2 by siRNA or by pharmacological inhibitor restored Notch signaling in myeloid cells and substantially improved their differentiation, both in vitro and in vivo. This study demonstrates a novel mechanism regulation of Notch signaling in cancer. This may suggest a new perspective for pharmacological regulation of differentiation of myeloid cells in cancer.

Figure 1. Down-regulation of Notch signaling in myeloid cells

A, B. Expression of hes1 (A) and hes5 (B) in CD34⁺ BM cells, Gr-1⁺ or CD11c⁺ splenocytes isolated from naïve or TB mice by qPCR. Cumulative results of 4 experiments are shown. * - statistically significant differences between groups (p<0.05). C. Expression of hes1 and hes5 in BM M-MDSC and PMN-MDSC from EL-4 TB mice and BM Mon and PMN from naive mice (n=3, * - statistically significant differences between groups (p<0.05)). D. CSL EMSA assay of nuclear proteins from enriched HPCs (naïve and indicated TB mice). Two experiments with the same results were performed. E. CSL/CBF1 reporter activity in HPCs from BM of naïve or CT26 TB mice. Cells were infected with CSL-IL-2-Luc or control KA9 viruses, and cultured on monolayer of 3T3-Jag1 expressing fibroblasts or control MSCV fibroblasts. Three experiments (each in duplicates) were performed. Significant differences between groups: * - p<0.05; ** - p<0.001. F. CSL reporter assay in Gr-1⁺ cells isolated from spleen of naïve or CT26 TB mice. Three experiments were performed. ** - p<0.01 between groups. G. Expression of HES1 in human cells isolated from patients with renal cells carcinoma and healthy donors. Four samples were analyzed by qPCR. Statistically significant differences between groups: *-p<0.05; **-p<0.01. H. Effect of indicated TCM on the expression of HES1 in CD34⁺ cells isolated from blood of healthy donors. Cells were analyzed after 3 days of culture. Three experiments were performed **-p<0.01 from control.

Figure 2. The role of Notch receptors and ligands in down regulation of Notch signaling

A. Nuclear translocation of Notch1 and Notch2. Nuclear proteins were analyzed by Western blot in HPCs from naïve or TB mice. Two experiments with the same results were performed. B, C. Expression of Notch1 (B) and Notch2 (C) in indicated cells isolated from naïve or CT26 TB mice and assessed by qPCR. Each experiment was performed in triplicate and includes 3 mice. * - statistically significant difference from control (p<0.05). D. Expression of notch1, notch2, and hes1 in indicated cells generated in vitro from HPCs. Each experiment was performed in triplicate and includes 3 mice. E. Notch ligands in BM of naïve and CT26 TB mice. Two experiments with the same results were performed. F. Expression of hes1 evaluated by qPCR in HPCs cultured on immobilized Dll1 with and without TCM. Each experiment was performed in triplicates. Cumulative results of three experiments are shown. G. Luciferase reporter assay in HPC cultured on Dll1 with and without TCM. pKA9 –control luciferase virus; pKA9-CBF1 – virus containing CSL binding site. Each experiment was performed in duplicate. Combined results of three experiments are shown. In all experiments: * - statistically significant difference from control (p<0.05).

Figure 3. Activation of Notch signaling was not able to rescue inhibited DC differentiation in TB mice

A, B. Differentiation of myeloid cells from HPC in the presence of immobilized Dll1 and TCM. Cells were cultured for 7 days and then phenotype (A) and function (B) (allogeneic MLR) was evaluated. Three experiments were performed. C. Differentiation of myeloid cells from HPC after transduction with ICN1. HPCs were infected with control (GFP) or ICN1-IRES-GFP (ICN) viruses and cultured with GM-CSF with (TCM) or without (Con) TCM for 7 days. Three experiments were performed. D, E. Differentiation of myeloid cells in vivo. Lin⁻c-kit⁺ HPCs from CD45.2 naïve BM were infected with control (GFP) or ICN1-IRES-GFP (ICN) viruses and injected i.v. to lethally irradiated CD45.1 naïve (N) or EL4 TB (TB) mice together with CD45.1⁺ BM cells. On day 14 cell phenotype was analyzed within population of GFP⁺CD45.2⁺ transduced donors cells. D. – typical example of the analysis. E. Cumulative results of three experiments. In all experiments: * - statistically significant difference from control (p<0.05).

Figure 4. Disruption of interaction between CSL and Notch and CK2 activity in myeloid cells

A–C. HPCs were transduced with ICN1-Myc virus. Co-precipitation and serine phosphorylation of ICN1 (A), ICN-Myc virus with 2 mutations (B) or 4 mutations (C) as described in methods. Cells were cultured with control media (CM) or TCM. On day 4, cells were collected and whole cell lysates were used for immunopreciptation with Myc antibody or control IgG and then probed with CSL antibody or phosphor-serine antibody as indicated. Each experiment was performed at least twice. D. The amount of CK2α in Gr-1⁺ cells from BM of naïve or TB mice. E, F. CK2 activity in HPC (E) or Gr-1⁺ cells (F) from BM of naïve or TB mice. Cumulative results of four performed experiments are shown. G. CK2 activity in Gr-1⁺ cells generated from HPCs after 5 day culture with control or TCM containing media. Cumulative results of three performed experiments are shown. H. CK2 activity in human CD11b⁺CD15⁺ cells isolated from blood of renal cell carcinoma patients and healthy donors (n=3). In all experiments statistically significant difference from control: * - p<0.05; ** - p<0.01.

Figure 5. CK2 regulates Notch activity and inhibits DC differentiation

A, B. 293T cells were co-transfected with CK2 plasmid pZW6 and pcDNA3.1-ICN-Myc plasmid. Expression of CK2α (A) and ICN1 serine phosphorylation (B) after immunoprecipitation with myc antibody was measured 48 hr later by Western blotting. Two experiments with the same results were performed. C. Phenotypes of DC differentiated from HPC transduced with CK2. Cells were analyzed on day 7 after infection with control or CK2 retroviruses and GFP⁺ cells were evaluated. Two experiments with the same results were performed. (D, E). Hes1 expression in MDSC transfected with CK2α siRNA. D - confirmation of down-regulation of CK2 expression using two different sets of siRNA. E. Hes1 expression. Cumulative results of three experiments are shown. MDSC were isolated from spleen of TB mice, transfected with scrambled or CKIIα siRNA, Hes1 expression was evaluated 24h later (D). ** - statistically significant difference from control (p<0.01).

Figure 6. Improvement of Notch signaling and DC differentiation by CK2 inhibitor

A, B. MDSC from spleen of TB mice were cultured for 48 hr with control or TCM containing medium in the presence of indicated concentrations of TBCA. CK2 activity (A) and serine phosphorylation of ICN (B) were assessed. C. HPC were cultured for 7 days with GM-CSF on immobilized Dll1 or control IgG in medium alone or in the presence of TCM. TBCA (2.5 μM) (CK2-I) was added at the start of the culture. Medium was replaced every other day. Cell phenotype was analyzed by flow cytometry. Results of three experiments are shown. * - statistically significant difference from control (p<0.01). D. HPC were cultured for 6 days with GM-CSF on immobilized Dll1 or control IgG in medium alone or in the presence of TCM. TBCA (5 μM) (CK2-I) was added at the start of the culture. Medium was replaced every other day. The phenotype of cells is shown. PMN-MDSC – Ly6C^loLy6G⁺ cells; M-MDSC – Ly6C^hiLy6G⁻ cells. Two experiments with the same results are shown.

Figure 7. Effect of CK2 inhibition on DC differentiation in vivo

CT26 TB mice were treated with TBCA (1.7 mg in 200 μl DMSO/water (1:4), i.p. daily) or vehicle alone (Con) for 6 days. Cells were collected on day 7. Each experiment include 4 mice. A. Tumor size in treated mice; B. CK2 activity of MDSC isolated from BM and spleens; C. Expression of hes1 mRNA in the same cells; D. Proportion of myeloid cells in spleens and lymph nodes. E, F. Absolute number of cells in spleens (E) and lymph nodes (F). G. Allogeneic MPR of splenocytes from treated mice. T cells from C57BL/6 mice were used as responder cells. In all experiments * indicated p<0.05 from control. H. EG-7 tumors were established s.c. by injecting 0.5×10⁶ cells. TBCA treatment started on day 7 after tumor inoculation and was continued for 14 days. Tumor size was measured. Each group included 3 mice.