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Genomic instability and immune evasion are hallmarks of cancer. Apoptotic cancer stem cells can evade cell death by undergoing cellular transformation by constructing “blebbishields” from apoptotic bodies. In this study, we report a novel linkage between genomic instability and phagocytosis evasion that is coordinated by the blebbishield emergency program. Blebbishield emergency program evaded genomic instability checkpoint, expressed genomic instability–associated genes at distinct phases of cellular transformation, exhibited chromosomal instability, and promoted increase in nuclear size. Blebbishields fused with immune cells to evade phagocytosis, and the resultant hybrid cells exhibited increased migration, tumorigenesis, metastasis, red blood cell recruitment to tumors, and induced hepatosplenomegaly with signatures of genomic instability, blebbishield emergency program, and phagocytosis evasion to offer poor prognosis. Overall, our data demonstrate that the blebbishield emergency program drives evasion of chromosomal instability and phagocytosis checkpoints by apoptotic cancer stem cells. Cancer Res; 77(22); 6144–56. ©2017 AACR.

Cellular transformation and the accumulation of genomic instability are the two key events required for tumorigenesis. K-Ras (Kirsten-rat sarcoma viral oncogene homolog) is a prominent oncogene that has been proven to drive tumorigenesis. K-Ras also modulates numerous genetic regulatory mechanisms and forms a large tumorigenesis network. In this review, we track the genetic aspects of K-Ras signaling networks and assemble the sequence of cellular events that constitute the tumorigenesis process, such as regulation of K-Ras expression (which is influenced by miRNA, small nucleolar RNA and lncRNA), activation of K-Ras (mutations), generation of reactive oxygen species (ROS), induction of DNA damage and apoptosis, induction of DNA damage repair pathways and ROS detoxification systems, cellular transformation after apoptosis by the blebbishield emergency program and the accumulation of genomic/chromosomal instability that leads to tumorigenesis.

Cancer stem cells are capable of undergoing cellular transformation after commencement of apoptosis through the blebbishield emergency program in a VEGF-VEGFR2-dependent manner. Development of therapeutics targeting the blebbishield emergency program would thus be important in cancer therapy. Specificity protein 1 (Sp1) orchestrates the transcription of both VEGF and VEGFR2; hence, Sp1 could act as a therapeutic target. Here, we demonstrate that CF3DODA-Me induced apoptosis, degraded Sp1, inhibited the expression of multiple drivers of the blebbishield emergency program such as VEGFR2, p70S6K, and N-Myc through activation of caspase-3, inhibited reactive oxygen species; and inhibited K-Ras activation to abolish transformation from blebbishields as well as transformation in soft agar. These findings confirm CF3DODA-Me as a potential therapeutic candidate that can induce apoptosis and block transformation from blebbishields.

Cancer stem cells are capable of transformation after apoptosis through the blebbishield emergency program. Reactive oxygen species (ROS) play an essential role in transformation. Understanding how ROS are linked to blebbishield-mediated transformation is necessary to develop efficient therapeutics that target the resurrection of cancer stem cells. Here we demonstrate that a novel PKC-ζ to p47phox interaction is required for ROS production in cancer cells. The combined use of the S6K inhibitor BI-D1870 with TNF-α inhibited the PKC-ζ to p47phox interaction, inhibited ROS production, degraded PKC-ζ and activated caspases-3 and -8 to block transformation from blebbishields. BI-D1870 also inhibited transformation from cycloheximide-generated blebbishields. Thus ROS and the PKC-ζ to p47phox interaction are valid therapeutic targets to block transformation from blebbishields.

The blebbishield emergency program helps to resurrect apoptotic cancer stem cells (CSCs) themselves. Understanding the mechanisms behind this program is essential to block resurrection of CSCs during cancer therapy. Here we demonstrate that endocytosis drives serpentine filopodia to construct blebbishields from apoptotic bodies and that a VEGF-VEGFR2-endocytosis-p70S6K axis governs subsequent transformation. Disengagement of RalGDS from E-cadherin initiates endocytosis of RalGDS and its novel interaction partners cdc42, VEGFR2, cleaved β-catenin, and PKC-ζ as well as its known interaction partner K-Ras. We also report novel interactions of p45S6K (cleaved p70S6K) and PKM-ζ with PAK-1 filopodia-forming machinery specifically in blebbishields. Thus, a RalGDS-endocytosis-filopodia-VEGFR2-K-Ras-p70S6K axis drives the blebbishield emergency program, and therapeutic targeting of this axis might prevent resurrection of CSCs during cancer therapy.

Background: Bleb transformation is an initial induction of apoptosis leading to cellular compartmentalization into vesicles, increased glycolysis/mitochondrial activity combined with dynamin mediated endocytosis allowing the cell to tether vesicles to form stem like spheroids protecting exposed genetic material. Our laboratory has found that not only do both Erlotinib sensitive and resistant cell lines undergo blebbishield transformation in response to treatment, but the addition of Glycyrrhetinic acid derivative (CDODA-Me) overwhelms this pathway leading to cellular apoptosis. Methods: NSCLC cell lines HCC827 (both erlotinib resistant and sensitive), were treated with CDODA-Me and Erlotinib (combination and single treatment). MicroRNA expression profiling was used to identify and validate reference genes involved with the synergistic effects comparing combination treatment to erlotinib single treatment groups. Spheroids were collected after 24hr treatment using blebbishield extraction media (BEM) consisting of 12µM erlotinib alone and in combination with 2µM CDODA-Me. Western Blot analysis was used to confirm identified pathways as well as to quantify the effects of CDODA-Me on VEGF and TGF-β cellular release levels during treatment as well as MET and EGFR kinase activation. Results: Genomic studies showed an increase in microRNA has-miR-711 (approximately 4-fold) which is responsible for inhibiting dynamin mediated trafficking, various growth factor expression levels (including VEGF and TGF-β), as well as the expression of receptors associated with these growth factors (EGFR, MET, VEGFR). This inhibition was confirmed through western blot analysis showing a decrease in receptor activation but no effects on receptor expression (6 fold and 8 fold decrease when compared to erlotinib single treatment for EGFR and MET respectively). Sphere reattachment was found to be dependent on the presence of growth factors and other unknown cell signaling molecules through the fact that spheres did not attach in fresh media, but attached in as little as 3hrs in media obtained after 24hr incubation in parental cells. Media obtained after incubation in parental cells provided higher levels of VEGF and TGF-β when compared to media obtained after 24hr treatment with (resistant to non-resistant expression ratios of 3.94 and 4.06 for HCC827 (4µM erlotinib resistant) and HCC827 Cl4 respectively). Actin fluorescent staining 48hrs after reseeding spheroids showed that cells grown in untreated parental media regained normal branch like morphology while those grown in CDODA-Me treated parental media remained rounded and died. Conclusions: Based on the results of these studies, CDODA-Me has been shown to inhibit NSCLC cell types from overcoming apoptosis thereby inhibiting increased drug resistance as well as tumorgenicity. Citation Format: Ebony Nottingham, Elizabeth Mazzio, Arindam Mondal, Mandip Sachdeva. The use of glycyrrhetinic acid derivatives to inhibit blebbishield emergency program pathogenesis overcoming erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4893.

Cancer stem cells evade apoptotic death by blebbishield emergency program, which constructs blebbishields from apoptotic bodies and drives cellular transformation. Von Hippel–Lindau (VHL) plays both tumor suppressor and oncogenic roles, and the reason behind is poorly understood. Here we demonstrate that dimers and trimers of p19-VHL interact with RalBP1 to construct blebbishields. Expression of RalBP1, p19-VHL, and high-molecular weight VHL is required to evade apoptosis by blebbishield-mediated transformation. In contrast, p30-VHL plays a tumor suppressor role by inhibiting blebbishield-mediated transformation. Furthermore, target genes of VHL that suppress oxidative stress were elevated during blebbishield-mediated cellular transformation. Thus, RalBP1 and p19-VHL play an oncogenic role, whereas p30-VHL plays a tumor suppressor role during the blebbishield emergency program by regulating oxidative stress management genes.

Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program.