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.

Aspirin inhibits proliferation and promotes differentiation of neuroblastoma cells via p21Waf1 protein up‐regulation and Rb1 pathway modulation

“…The inhibitor of apoptosis protein (IAP) survivin is highly up‐regulated in many transformed cell lines including lung, colon, breast and brain human cancers, where it promotes proliferation, invasiveness and inhibition of apoptosis, the latter in particular conferring resistance to chemotherapy. Therefore, elevated levels of this protein correlate with poor prognosis and radio/chemo‐resistance of several cancers, including NB . Here, we report that aspirin time dependently decreased the survivin expression, whose levels almost disappeared after 5 days.…”

The Role of Rho GTPases in VEGF Signaling in Cancer Cells

“…Finally, we noticed the complexity and interconnectedness between VEGF and Rho-related pathways which benefit cancer cells in multiple aspects which include but are not limited to angiogenesis, migration, and invasion. Due to the complications associated with the untargeted cancer therapies (chemo-and radiotherapy), approaches targeting specific characteristics and pathways of tumors must be explored [80,[125][126][127][128][129]. Therefore, targeting these interaction factors that are produced and secreted by the tumor cells might exhibit promising therapeutic potential.…”

Mesenchymal Epithelial Transition Factor Signaling in Pediatric Nervous System Tumors: Implications for Malignancy and Cancer Stem Cell Enrichment

“…Neuroblastoma is the most common extracranial solid tumor in children, which arises from the neural crest cells, destined to differentiate into the sympathetic chain ganglia and adrenal medulla, which are components of the sympathetic nervous system ( Abou-Antoun et al, 2018 ). NB presents with a wide clinical heterogeneity, where in some cases, it undergoes spontaneous regression, whereas in other cases, it progresses aggressively leading to mortality ( Chakrabarti et al, 2012 ).…”