Vascular development and safeguard mechanisms against tumorigenesi s: oncogene-induced apoptosis and cellular senescence

Angiogenesis, apoptosis and senescence are all cellular processes that have an impact on tumor development. Angiogenesis or the new vessel formation from pre-existing ones is known to be required for invasive tumor growth and metastasis. Apoptosis and cellular senescence are both considered crucial safeguards mechanisms against neoplastic transformation. The MYC oncogene plays an important role in the regulation of all three of these as well as many other fundamental processes crucial for cell growth and tumorigenesis. In the first part of this thesis we explored endothelial cell migration by the exposure of human vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) to stable hill-shaped gradients of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). Time-lapse analysis showed that a gradient of VEGFA165 efficiently induced chemotaxis of endothelial cells of different vascular origin. Stable gradients of FGF2 were able to attract venular but no arterial endothelial cells. In addition to the directed migration of endothelial cells, we also investigated the lymphatic vessel formation in the developing mice kidney. Immunohistochemical analysis of kidney explants and whole mount of dissected kidney suggested that renal lymphatic vessel formation predominately occurs via invasive sprouting from surrounding lymphatic plexus. In the second part of this thesis, we first aimed to clarify the relative importance of the intrinsic (mitochondrial) and extrinsic (death receptor) anti apoptotic pathways in the in vivo MYC driven transformation of hematopoietic stem cells. Expression of MYC alone resulted in the development of both myeloid and T-lymphoid tumors within two months after transplantation of HSCs. Expression of MYC together with BCL-XL or BCL-2 (inhibiting the intrinsic pathway) resulted in almost immediate development of AML like disease. In contrast, expression of MYC together with FLIPL (inhibiting the extrinsic pathway) did not accelerate tumorigenesis. These results suggest that MYC-induced transformation of HSC accelerates and polarizes hematopoietic tumor development towards aggressive AML by co-expression of inhibitors of the intrinsic but not the extrinsic pathway of apoptosis. Secondly, we aimed to determine whether pharmacological inhibition of cyclin dependent kinase 2 (CDK2) interferes with MYC-driven tumor development in vivo trough senescence. Mice transplanted with HSCs expressing MYC and BCL-XL as briefly described above were treated with a specific CDK2 inhibitor on daily basis via intraperitoneal injections or osmotic minipumps. Despite the very aggressive AML development in this model, CDK2 targeting significantly delayed the onset of disease and improved mice survival by restoring senescence. The senescence induction correlated with induction of p19ARF, p21CIP1 and activation of pRb. This suggests that pro-senescence therapy via CDK2 inhibition should be further evaluated as a new potential strategy to combat MYC-driven AML and possibly other MYC-related tumors

Inhibition of the Intrinsic but Not the Extrinsic Apoptosis Pathway Accelerates and Drives Myc-Driven Tumorigenesis Towards Acute Myeloid Leukemia

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-XL/BCL-2 (inhibiting the intrinsic pathway) or FLIPL (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7–9 weeks as expected. Importantly, coexpression of MYC together with BCL-XL/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIPL did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4+CD8+ versus mature CD4+ T-cell lymphoma was observed in MYC/FLIPL mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-XL and BCL-2 but not FLIPL acts in synergy with MYC to drive AML development

Tumor phenotype in Mock/MYC, FLIP_L/MYC, BCL-X_L/MYC and BCL-2/MYC recipient mice.

<p>Summary of flow cytometry analysis of spleen cells from moribund mice transplanted with HSC expressing (A) Mock-GFP/MYC-YFP into DBA/2 mice, (B) FLIP_L-GFP/MYC-YFP into DBA/2 mice, (C) BCL-X_L-GFP/MYC-YFP into DBA2 mice, (D) BCL-X_L-GFP/MYC-YFP into BALB/c mice and (E) BCL-2-GFP/MYC-YFP into BALB/c mice. Left panel specify tumors expressing MYC-YFP only and right panel specify cells expressing MYC-YFP together with either Mock-GFP (A), FLIP_L-GFP (B), BCL-X_L-GFP (C and D) or BCL-2-GFP (E). Filled box indicate tumor phenotype. M indicates tumors of myeloid lineage, DP indicate CD4⁺CD8⁺ lymphoid tumors, 4 indicate CD4⁺ lymphoid tumors and 8 indicate CD8⁺ lymphoid tumors. Numbers correspond to identity of individual animal.</p

Overexpression of MYC in HSC induces both myeloid and lymphoid leukemia.

<p>Flow cytometry analysis of bone marrow and spleen cells from two individual moribund Mock/MYC mice (indicated with # in the left). In the middle, percentage of GFP⁺YFP⁺ (Mock/MYC expressing) cells or GFP⁻YFP⁺ (MYC expressing) cells are indicated. These cells were further characterized with anti-CD11b, anti-Gr1, anti-CD4, anti-CD8, anti-CD19, anti-IgM anti-CD71 and anti-Ter119. The percentage of cells in each quadrant is indicated.</p

Overexpression of BCL-X_L-GFP/MYC-YFP induces AML.

<p>Flow cytometry analysis of bone marrow (top), spleen (middle) and liver (bottom) cells from one representative moribund BCL-X_L-GFP/MYC-YFP mouse. Single cell suspensions from femoral bone marrow, spleen and liver were stained with anti-CD11b and anti-Gr1. The percentage of dominating cells is indicated.</p

BCL-X_L and BCL-2 but not FLIP_L accelerate Myc-induced hematopoietic tumorigenesis.

<p>Kaplan-Meier survival analysis of mice transplanted with HSC over-expressing (A) Mock-GFP/MYC-YFP in DBA/2, (B) BCL-X_L -GFP/MYC-YFP in DBA/2, (C) FLIP_L-GFP/MYC-YFP in DBA/2, (D) BCL-X_L-GFP/MYC-YFP in BALB/c and (E) BCL-2-GFP/MYC-YFP in BALB/c. Mice were monitored daily for tumors or signs of paralysis and killed if showing signs of sickness. The percentage of mice surviving at daily intervals is shown.</p

Over-expression of BCL-X_L and BCL-2 accelerate Myc-induced splenomegaly whereas co-expression of FLIP_L does not influence MYC-induced splenomegaly.

<p>Spleen weight of animals transplanted with HSC expressing (A) Mock-GFP/Mock-YFP into DBA/2 mice, (B) Mock-GFP/MYC-YFP into DBA/2 mice, (C) FLIP_L-GFP/MYC-YFP into DBA/2 mice, (D) BCL-X_L-GFP/MYC-YFP into DBA2 mice, (E) BCL-X_L-GFP/MYC-YFP into BALB/c mice and (F) BCL-2-GFP/MYC-YFP into BALB/c. Spleens of mice at control time points (7 days, 14 days, 35 days and 49 days after transplantation) and spleens of all moribund mice were weighed. Each dot represents one mouse.</p

Blast formation of Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP or BCL-X_L-GFP/MYC-YFP bone marrow and spleen cells.

<p>(A) Forward light scatter of CD11b⁺Gr1⁺ bone marrow cells from Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP and BCL-X_L-GFP/MYC-YFP recipient mice 2 weeks after transplantation. Grey thick lines indicate GFP⁻YFP⁺ cells, thick black lines indicate GFP⁺YFP⁻ cells and thin lines indicate GFP⁺YFP⁺ cells. (B) Difference in mean fluorescence intensity (Δmfi) in forward scatter of CD11b⁺Gr1⁺ (left), CD19⁺IgM⁻ (middle) and CD19⁺IgM⁺ (right) cells in bone marrow (top) and spleen (bottom) between GFP⁻YFP⁻ (non-transduced cells) and GFP⁻YFP⁺ (black bars), GFP⁺YFP⁺ (striped bars) or GFP⁺YFP⁻ (white bars) cells are shown. Values indicate means of three individual mice and error bars indicate 1 SD.</p

Statistical analysis of survival data after transplantation of hematopoietic stem cells expressing MYC, BCL-X_L, BCL-2 and/or FLIP_L.

<p>Statistical analysis of the survival data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031366#pone-0031366-g002" target="_blank">Figure 2</a>.</p>*<p>Log-rank (Mantel-Cox) test. NS = non significant. S = significant. P-value indicated below.</p

Expression of the various combinations of genes in Lin⁻ cells prior to transplantation.

<p>FACS analysis of expression of YFP and GFP in HSC prior to transplantation in HSC derived from (A) DBA/2 mice transduced with Mock-GFP and Mock-YFP, (B) DBA/2 mice transduced with Mock-GFP and MYC-YFP, (C) DBA/2 mice transduced with BCL-X_L-GFP and MYC-YFP, (D) DBA/2 mice transduced with FLIP_L-GFP and MYC-YFP, (E) BALB/c mice transduced with BCL-X_L-GFP or MYC-YFP and (F) BALB/c mice transduced with BCL-2-GFP and MYC-YFP. The percentage of cells expressing the genes in each quadrant, gated on PI negative cells, is indicated.</p