Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance

Abstract

Despite significant advances in cancer treatment and management, more than 60% of patients with neuroblastoma present with very poor prognosis in the form of metastatic and aggressive disease. Solid tumors including neuroblastoma are thought to be heterogeneous with a sub-population of stem-like cells that are treatment-evasive with highly malignant characteristics. We previously identified a phenomenon of reversible adaptive plasticity (RAP) between anchorage dependent (AD) cells and anchorage independent (AI) tumorspheres in neuroblastoma cell cultures. To expand our molecular characterization of the AI tumorspheres, we sought to define the comprehensive proteomic profile of murine AD and AI neuroblastoma cells. The proteomic profiles of the two phenotypic cell populations were compared to each other to determine the differential protein expression and molecular pathways of interest. We report exclusive or significant up-regulation of tumorigenic pathways expressed by the AI tumorspheres compared to the AD cancer cells. These pathways govern metastatic potential, enhanced malignancy and epithelial to mesenchymal transition. Furthermore, radio-therapy induced significant up-regulation of specific tumorigenic and proliferative proteins, namely survivin, CDC2 and the enzyme Poly [ADP-ribose] polymerase 1. Bio-functional characteristics of the AI tumorspheres were resistant to sutent inhibition of receptor tyrosine kinases (RTKs) as well as to 2.5 Gy radio-therapy as assessed by cell survival, proliferation, apoptosis and migration. Interestingly, PDGF-BB stimulation of the PDGFRβ led to transactivation of EGFR and VEGFR in AI tumorspheres more potently than in AD cells. Sutent inhibition of PDGFRβ abrogated this transactivation in both cell types. In addition, 48 h sutent treatment significantly down-regulated the protein expression of PDGFRβ, MYCN, SOX2 and Survivin in the AI tumorspheres and inhibited tumorsphere self-renewal. Radio-sensitivity in AI tumorspheres was enhanced when sutent treatment was combined with survivin knock-down. We conclude that AI tumorspheres have a differential protein expression compared to AD cancer cells that contribute to their malignant phenotype and radio-resistance. Specific targeting of both cellular phenotypes is needed to improve outcomes in neuroblastoma patients.

Fig 1. Proteomic profile analysis reveals functional categories of proteins differentially expressed between AI tumorspheres and AD neuro2a cells.

(A) Compared to NB AD adhered cells, AI tumorspheres are comprised of proteins that are highly involved in small molecule biochemistry, RNA post translational modification and energy production and metabolism. (B) String Functional Protein pathway analysis revealed affiliations between the tumorigenic proteins in the AI tumorspheres, specifically highlighted are HDGFR and RhoA pathways and their affiliated interactions with other tumorigenic proteins. The HDGFR affiliated proteins are involved in mitogenic and DNA-binding activity and may play roles in cellular proliferation and differentiation and histone methylation; whereas the RhoA affiliated proteins play key roles in the GDP-GTP exchange and activation of Rho-GTPs involved in cell adhesion, migration and invasion.

Fig 2. Western blot validation of specific proteins identified in the proteomic analysis.

Western blot analysis was used to validate some of the targets identified on proteomic profiling. The protein expression of CDC2 was 2-fold higher in the AD cells compared to the AI tumorspheres, and PARP1, SOX2 and HDGFR were expressed > 1.5-fold higher in the AI tumorspheres compared to the AD cells. In addition, the expression levels of MIA3, RhoA, PDGFRβ, VEGFR and EGFR was also significantly (P < 0.05) higher in the AI tumorspheres, as determined with Western blot analysis. * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 3. AI tumorspheres exhibit resistance to radio-therapy compared to AD cancer cells.

(A) AD cells (black bars) had a significantly (*P < 0.05) higher rate of apoptosis 2 days after a single dose (2.5 Gy) of radiation compared to AI tumorspheres (white bars) as assessed using 7AAD/Annexin V staining. (B) AD cells had a more potent reduction (81% to 38%) in the proliferation marker Ki-67 expression than AI tumorspheres (72% to 52%) at 72 h post radiation compared to control cells. The average ratio of Ki-67% irradiated/control cells in the AI tumorspheres was 0.75 whereas that of the AD cells was 0.45, as derived from the mean of 4 independent experiments run in triplicates and presented ±SEM of multiple experiments. (C) AI tumorspheres (left side) exhibited a higher number of viable cells over 96 h post radiation compared to AD cells (right side) as determined by counting viable (non-trypan blue stained) and non-viable (trypan blue stained) cells, used in support of the XTT cell proliferation assay. (D) The average ratio of irradiated/control neuroblastoma AD cells (black bars) and that of AI tumorspheres (white bars) was derived from the absorbance of 4 independent experiments run in triplicates and graphed. The ratio was significantly different (*P < 0.05) between the AD cells and AI tumorspheres at 48, 72 and 96 h post radio-therapy(2.5 Gy) indicating the radio-resistant nature of the AI tumorspheres compared to the AD cells. (E) AD cells exhibit increased cell death after radio-therapy compared to AI tumorspheres as shown in the photographic representation of the AI tumorspheres and AD Cells 72 h post radio-therapy revealing more dead cells and cellular debris in the AD cells compared to the AI tumorspheres. * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 4. AI tumorspheres and AD cells exhibit significant up-regulation of survivin, PARP 1 and CDC2 post radiation.

AI tumorspheres or AD cells were either irradiated (+) (2.5 Gy) or left un-treated (-) and cells were lysed after 48 h of irradiation and proteins separated on a SDS-page gel for Western blot analysis. Survivin was significantly up-regulated after irradiation of both AI tumorspheres and AD cells compared to untreated controls. Furthermore, PARP 1 was significantly up-regulated after irradiation of both AI tumorspheres and AD cells compared to untreated controls. In addition, CDC2, a proliferative marker, which was significantly over-expressed by the AD cells compared to the AI tumorspheres, showed significant up-regulation in both AI tumorspheres and AD cells 48 h post irradiation compared to untreated cells. * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 5. Small molecule inhibition of the PDGFRβ using sutent significantly increases cell death and inhibits cell proliferation and migration in AD cells.

(A) NB AI tumorspheres (white bars) exhibited significant resistance to increasing concentrations of sutent treatment compared to un-treated counterparts, as assessed using a cytotoxicity assay after 48 h of treatment, whereas AD cells (black bars) showed a dose-dependent increase in cell death, compared to un-treated counterparts. (B) The average ratio of treated/control absorbance of NB AI tumorspheres and AD cells from four independent experiments run in triplicates was derived and graphed ± SEM. The AI tumorspheres (white bars) were significantly resistant to sutent-induced reduction in cell proliferation compared to their un-treated counterparts over 24, 48, 72 and 96 h post treatment, whereas AD cells (Black bars) showed a marked reduction in cell proliferation after sutent treatment compared to their un-treated counterparts as determined using a WST-1 cell proliferation assay. (C) A “wound-healing” assay was used to assess the PDGF-BB (10 ng) induced cell migration of NB AD cells, treated with sutent for one hour before ‘wound-induction’. Sutent significantly inhibited the AD cells’ ability to migrate into and close the wound after 24 h. (D) A trans-well chemotactic migration chamber was used to measure the PDGF-BB induced cell migration of AD and AI cells that migrated to the underside of the chamber with or without sutent treatment. The average absorbance was represented as a ‘fold-change’ between the groups; unstimulated, untreated cells (Control) were normalized to 1, whereas PDGF-BB induced migration (control + BB) and sutent-induced inhibition of PDGF-BB stimulated migration (sutent + BB) was measured as a fold-change. PDGF-BB stimulation led to a significant (*) increase in AI (white bars) and AD (Grey bars) cell migration compared to their un-stimulated controls. Sutent treatment significantly (#) abrogated (P < 0.05) the PDGF-BB induced AD cells’ (grey bars) ability to migrate to the lower side of the chamber (Fold-change from 1.65 to 1.2), but had only a slight, but not significant (fold-change from 1.75 to 1.65) effect on the trans-well migration capacity of AI tumorspheres (white bars). * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 6. Sutent inhibits the phosphorylation of the PDGFRβ and EGFR/VEGFR trans-activation.

(A) Western blot analysis of AI tumorspheres compared to NB AD cells showed significant phosphorylation (B) of PDGFRβ and the downstream signaling cascade (Akt) after 5 mins stimulation with 10 ng PDGF-BB in AI tumorspheres, which was significantly inhibited after 1 h of 0.2 μM sutent pre-treatment. AD cells were less-affected by PDGF-BB stimulation compared to the AI tumorspheres (A and B). In addition, AD and more potently AI cells stimulated with PDGF-BB for 5 minutes exhibited trans-activation of both EGFR and VEGFR that was abolished with sutent pre-treatment for 1 h (A and B). * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 7. Sutent combined with survivin knock-down exhibits added effect on inhibition of cell migration and invasion and enhanced AI radio-sensitivity.

(A) NB AD cells treated with 0.2 μM sutent plus survivin knock-down exhibited an added effect on the inhibition of cell migration as assessed with a ‘wound-healing’ assay compared to either treatment alone. (B) AI tumorspheres (white bars) treated with a combination of sutent (0.2 μM) and survivin knock-down exhibited a significant reduction in PDGF-BB (+) mediated cell invasion capacity compared to sutent treatment alone or untreated controls. AD cells (black bars) treated with sutent alone exhibited inhibition in PDGF-BB (+) mediated cell invasion, which was also enhanced in combination with survivin knock-down. (C) Survivin knock-down in AI tumorspheres increased radio-sensitivity when combined with sutent treatment by exhibiting an added effect on inhibition of cell proliferation after radio-therapy compared to survivin knock-down alone. (D) Western blot analysis confirms significant knock-down of survivin protein expression in neuro2a AD cells and AI tumorspheres measured after 48 h of siRNA transfection. * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.

Fig 8. Long-term sutent treatment in AI tumorspheres inhibits tumorigenic protein expression and tumorsphere self-renewal.

AI tumorspheres were treated with 0.2 μM sutent for 48 h and then lysed for Western blot analysis. A) Western blot images of representative experiments illustrating the reduced expression of PDGFRβ, MYCN, SOX2 and Survivin after 48 h sutent treatment. B) Densitometric analysis of multiple Western blot experiments showing statistically significant reduction in the protein expression of the above mentioned proteins. C) Micro-images of tumorsphere self-renewal capacity over 15-days, inhibited with 0.2 μM sutent treatment. * indicates a P value < 0.05. Results represent the mean ± SEM of multiple experiments.