Breaking the 'harmony' of TNF-α signaling for cancer treatment

Abstract

Tumor necrosis factor-alpha (TNF-α) binds to two distinct receptors, TNFR1/p55 and TNFR2/p75. TNF-α is implicated in the processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We have shown that TNFR2/p75 signaling promotes ischemia-induced angiogenesis via modulation of several angiogenic growth factors. We hypothesized that TNFR2/p75 may promote tumor growth and angiogenesis. Growth of mouse Lewis lung carcinoma (LLC1) and/or mouse melanoma B16 cell was evaluated in wild type (WT), p75 knockout (KO) and double p55KO/p75KO mouse tumor xenograft models. Compared with WT and p55KO/p75KO mice, growth of tumors in p75KO mice was significantly decreased (twofold) in both LLC and B16 tumors. Tumor growth inhibition was correlated with decreases in vascular endothelial growth factor (VEGF) expression and capillary density, as well as bone marrow-derived endothelial progenitor cells incorporation into the functional capillary network, and an increase in apoptotic cells in LLC xenografts. Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival. Blocking p75 by short-hairpin RNA in cultured LLCs led to increases in TNF-mediated apoptosis, as well as decreases in the constitutive and TNF-mediated expression of angiogenic growth factors (VEGF, HGF, PLGF), and SDF-1α receptor CXCR4. In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts. Blocking p75 expression may lead to tumor regression. This may represent new and effective therapy against lung neoplasms and potentially tumors of other origin.

Figure 1. Tumor growth inhibition in p75KO mice is associated with decreased expression of VEGF and capillary density, as well as increased apoptosis, while TNF expression is similar in WT and p75KO tumors

A, Graphic representation of LLC tumor volumes in WT, p75KO and Dbl-KO mice. Graphs represent pooled data from 3 independent experiments (N=18–24/treatment group). B, Quantification of TNF immunostaining (red) in tumors and normal skin of WT and p75KO mice shown as percent of mean pixel intensity using NIH Image J program (here and elsewhere). C, TNF protein release (pg/ml) measured in tumor homogenates from WT and p75KO mice (p=NS). D, Quantification of VEGF immunostaining (red) in tumors from WT and p75KO mice shown as percent of mean pixel intensity (p<0.001, WT vs. p75KO). E, VEGF protein release (pg/ml) measured in tumor homogenates (p<0.002, WT vs. p75KO). F–G, Representative images (right panel) and quantification of – F, CD31 (red) immunofluorescence, and G, Quantification of CD31 (+) cells in the whole tumor tissue from WT vs p75KO mice using FASC analysis, when WT is set as 100%. Please note, compared to FASC analysis, tumor associated CD31 (+) cells were twice as higher in immunofluorescent studies, suggesting a significant heterogeneity in tumor vascular network and a superior quantitative nature of the FACS analysis of tumor associated CD31 (+) cells. H, Representative images (right panel) and quantification of –H, TUNEL (green) immunostaining in WT and p75KO tumors, shown as percent of mean pixel intensity, when WT is set as 100%. Results in all graphs are pooled data (mean+SEM) from 3 independent experiments 7–8 fields of 176,400 μm² (image size here and elsewhere) per mouse, N= 5 mice/genotype.

Figure 2. Increased apoptosis in tumors from p75KO mice at the border-zone and reduced incorporation of BM-derived EPCs into functional capillary network in p75KO tumors

Apoptosis and tumor angiogenesis was also evaluated at the interface of tumor/normal tissue by triple staining with Terminal Transferase dUTP Nick End Labeling (TUNEL), CD31 and Topro-3. The peri-tumoral and tumor area was identified by H&E staining of adjacent sections (not shown). Representative images of triple-immunostained tumors (panel on the far right) in the periphery of tumor tissue for TUNEL (green), CD-31 (red) and Topro-3 (blue) in WT (A) and p75KO (B). A–B, TUNEL staining (top panel) in WT tumors. B, Insets in peri-tumoral (top) and tumors (bottom) in p75KOs show double positive (TUNEL/CD31-yellowish staining, arrowheads) indicating apoptosis of p75KO ECs. C, Representative images of BM-derived (GFP +) cells recruited into WT and p75KO tumor tissue (arrows). D, Quantification of BM-derived GFP (+) cells recruitment into the tumor tissue. E, Representative images of double BM-derived GFP (+)/BS-1/lectin (+) cells, incorporated into functional vessels (yellow staining, arrows). Small arrowheads indicate BM-derived GFP (+) cells that are not incorporated into functional vessels (green) in p75KO tumors. F, Quantification of BM-derived EPCs incorporation into the functional capillary network.

Figure 3. Angiogenic genes expression is decreased in tumor tissue from p75KO mice

A and B, Representative Angiogenesis pathways microarray of tumors from WT and p75KO mice at 5 minute exposure and the loading controls (bottom, small insets) at 5 second exposure time. Dotted-line circles indicate gene expression decrease in p75KO vs. the same gene in WT (solid-line circles). C, Functional grouping of genes in angiogenesis pathways microarray with the fold changes (dotted-line circles - decreased) in p75KO vs WT tumors. In p75KO tumor tissue decreased gene expression was observed in growth factors, cytokines and chemokines, such as - vascular endothelial growth factor (VEGFA and B), placental growth factor (PGF), chemokine (C-X-C motif) ligands (Cxcl1, Cxcl2, Cxcl10), interleukins (IL1b, IL12a and IL18), pleiotrophin (Ptn, known as, heparin-binding growth factor 8), signal transduction and transcription factors Mapk14 (know as p38), prostaglandin-endoperoxide synthase 1 (Ptgs1), adhesion molecules and proteases such as, collagen, type XVIII, alpha 1 (Col18a1), neuropilin 1 (Nrp1), angiopoietin-like 4 (Angptl4) and matrix metalloproteinase 9 – Mmp 9). The densitometry values of Hsp90ab1 and BAS2C genes were used as internal control. An arbitrary cut off was set at 1.6 (up or down). We assigned an arbitrary cut off thresholds for fold changes at 1.6 (here and elsewhere).

Figure 4. Anti-apoptotic gene expression is decreased in p75KO tumor tissue

A and B, Representative apoptosis pathways microarray of tumors from WT and p75KO mice at 5 minute exposure and the loading controls (bottom, small insets) at 5 second exposure time. Functional grouping of genes in apoptosis microarray with the fold changes of the same gene (solid-line vs. dotted-line circles – decreased) in WT vs p75KO.

Figure 5. Signaling through TNFR2/p75 is required for proper activation of p38 MAPK in EPCs

A, C, E, Representative images of Phospho-p38 (P-p38) and Total-p38 (T-p38) western blot analysis in WT and p75KO EPCs and unaltered LLCs. B, D, F, Quantification p38 protein level and phosphorylation using densitometric analysis of P-p38 band intensity after adjusting for actin (not shown) and T-p38 band intensity. Band intensity at time 0 for each cell type was set at 100% and percent change over 60 minutes was calculated. Results represent data from three independent experiments. Statistical significance was assigned when p<0.05.

Figure 6. Percent inhibition of p75 receptor expression correlates directly with increase in TNF-mediated apoptosis and decrease in angiogenic gene expression in p75KD/LLCs

Representative histograms of FACS analysis of propidium iodide (PI)-stained two p75KD/LLC (transfected with p75 shRNA plasmid combinations 1+2 and 1−4), where M1 gates represent sub-G0/G1 population of the cells with less than 2n DNA, presumably, apoptotic cells. B–E, Constitutive and TNF-mediated (80 ng/ml) expression (qRT-PCR analysis) for various angiogenic genes – B, VEGFA, C, HGF, C, PLGF and E, CXCR4 for the same two p75KD/LLCs 24 hrs post-TNF treatment.