Current ovarian cancer maintenance therapy is limited by toxicity and no proven impact on overall survival. To study a maintenance strategy targeted at missense mutant p53, we hypothesized that the release of mutant p53 from mortalin inhibition by the SHetA2 drug combined with reactivation of mutant p53 with the PRIMA‐1MET drug inhibits growth and tumor establishment synergistically in a mutant‐p53 dependent manner. The Cancer Genome Atlas (TCGA) data and serous ovarian tumors were evaluated for TP53 and HSPA9/mortalin status. SHetA2 and PRIMA‐1MET were tested in ovarian cancer cell lines and fallopian tube secretory epithelial cells using isobolograms, fluorescent cytometry, Western blots and ELISAs. Drugs were administered to mice after peritoneal injection of MESOV mutant p53 ovarian cancer cells and prior to tumor establishment, which was evaluated by logistic regression. Fifty‐eight percent of TP53 mutations were missense and there were no mortalin mutations in TCGA high‐grade serous ovarian cancers. Mortalin levels were sequentially increased in serous benign, borderline and carcinoma tumors. SHetA2 caused p53 nuclear and mitochondrial accumulation in cancer, but not in healthy, cells. Endogenous or exogenous mutant p53 increased SHetA2 resistance. PRIMA‐1MET decreased this resistance and interacted synergistically with SHetA2 in mutant and wild type p53‐expressing cell lines in association with elevated reactive oxygen species/ATP ratios. Tumor‐free rates in animals were 0% (controls), 25% (PRIMA1MET), 42% (SHetA2) and 67% (combination). SHetA2 (p = 0.004) and PRIMA1MET (p = 0.048) functioned additively in preventing tumor development with no observed toxicity. These results justify the development of SHetA2 and PRIMA‐1MET alone and in combination for ovarian cancer maintenance therapy.
Clinical outcomes for high-risk neuroblastoma patients remains poor, with only 40–50% 5-Year overall survival (OS) and <10% long-term survival. The ongoing acquisition of genetic/molecular rearrangements in undifferentiated neural crest cells may endorse neuroblastoma progression. This study recognized the loss of Retinal Degeneration protein 3, RD3 in aggressive neuroblastoma, and identified its influence in better clinical outcomes and defined its novel metastasis suppressor function. The results showed ubiquitous expression of RD3 in healthy tissues, complete-loss and significant TNM-stage association of RD3 in clinical samples. RD3-loss was intrinsically associated with reduced OS, abridged relapse-free survival, aggressive stage etc., in neuroblastoma patient cohorts. RD3 was transcriptionally and translationally regulated in metastatic site-derived aggressive (MSDAC) cells (regardless of CSC status) ex vivo and in tumor manifolds from metastatic sites in reproducible aggressive disease models in vivo. Re-expressing RD3 in MSDACs reverted their metastatic potential both in vitro and in vivo. Conversely muting RD3 in neuroblastoma cells not only heightened invasion/migration but also dictated aggressive disease with metastasis. These results demonstrate the loss of RD3 in high-risk neuroblastoma, its novel, thus-far unrecognized metastasis suppressor function and further imply that RD3-loss may directly relate to tumor aggressiveness and poor clinical outcomes.
Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.
IntroductionTherapy-associated onset of stemness-maintenance in surviving tumor-cells dictates tumor relapse/recurrence. Recently, we recognized the anti-pancreatic cancer (PC) potential of seaweed polyphenol manifolds and narrowed down three superior drug-deliverables that could serve as adjuvants and benefit PC cure. Utilizing the PC- cancer stem cells (PC-CSCs) grown ex vivo and mouse model of residual-PC, we investigated the benefits of seaweed polyphenols in regulating stemness-maintenance.MethodsALDH+CD44+CD24+ PC-CSCs from Panc-1, Panc-3.27, MiaPaCa-2, or BxPC-3 cells-derived xenografts grown ex vivo were either mock-irradiated, exposed to fractionated irradiation (FIR, 2Gy/D for 5 days), treated with polyphenols (100 μg/ml) of Hormophysa triquerta (HT-EA), Spatoglossum asperum (SA-EA) or Padina tetrastromatica (PT-EA) with/without FIR were examined for cell viability, transcription of 93 stem-cell-related molecules (QPCR profiling). Polyphenol-dependent regulation of FIR-transactivated Oct4, Zic3, EIF4C, Nanog, and LIF (QPCR) and functional translation of Nanog, SOX2, and OCT3/4 (immunoblotting) were examined in Panc-1/Panc-3.27/MiaPaCa-2/BxPC-3-xenografts derived PC-CSCs. Effect of seaweed-polyphenols in the regulation of EMT (N-Cadherin), pluripotency- (SOX2, OCT3/4, Nanog) and stemness-maintenance (PI3KR1, LIF, CD44) in therapy (FIR, 2Gy/D for 5D/wk for 3-weeks) resistant residual tumors were examined by tissue microarray construction and automated immunohistochemistry.ResultsEx vivo exposure of PC-CSCs to SA-EA, PT-EA and HT-EA exhibit dose-dependent inhibition of cell viability. FIR amplified the transcription of 69, 80, 74 and 77 stem-cell related genes in MiaPaCa-2-, Panc-1-, Panc-3.27- and BXPC3-established xenograft-derived ALDH+CD44+CD24+PC-CSCs. Treatment with SA-EA, PT-EA, or HT-EA completely suppressed FIR-activated stem-cell transcriptional machinery in ALDH+CD44+CD24+PC-CSCs established from MiaPaCa-2, Panc-1, Panc-3.27 and BXPC3 xenografts. QPCR validated EIF4C, OCT3/4, Nanog, LIF, and ZIC3 transcriptional profile outcomes. Nanog, Sox2, and OCT3/4 immunoblotting affirmed the PC-CSC radiosensitizing benefit of seaweed polyphenols. Residual-PC tissues microarrayed and immunostained after in vivo treatments recognized complete regulation of FIR-induced SOX2, OCT3/4, Nanog, LIF, CD44, PIK3R1, N-Cadherin, and E-Cadherin with SA-EA, PT-EA, and HT-EA.ConclusionsThese data, for the first time, documented the EMT/stemness-maintenance in therapy-resistant PC-CSCs. Further, the data suggest that seaweed polyphenols may inhibit PC relapse/recurrence by targeting therapy-orchestrated stem-cell signaling in residual cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0173-3) contains supplementary material, which is available to authorized users.
BackgroundCirculating miRNAs have momentous clinical relevance as prognostic biomarkers and in the progression of solid tumors. Recognizing novel candidates of neuroblastoma-specific circulating miRNAs would allow us to identify potential prognostic biomarkers that could predict the switch from favorable to high-risk metastatic neuroblastoma (HR-NB).ResultsUtilizing mouse models of favorable and HR-NB and whole miRnome profiling, we identified high serum levels of 34 and low levels of 46 miRNAs in animals with HR-NB. Preferential sequence homology exclusion of mouse miRNAs identified 25 (11 increased; 14 decreased) human-specific prognostic marker candidates, of which, 21 were unique to HR-NB. miRNA QPCR validated miRnome profile. Target analysis defined the candidate miRNAs' signal transduction flow-through and demonstrated their converged roles in tumor progression. miRNA silencing studies verified the function of select miRNAs on the translation of at least 14 target proteins. Expressions of critical targets that correlate tumor progression in tissue of multifarious organs identify the orchestration of HR-NB. Significant (>10 fold) increase in serum levels of miR-381, miR-548h, and miR-580 identify them as potential prognostic markers for neuroblastoma progression.ConclusionFor the first time, we identified serum-circulating miRNAs that predict the switch from favorable to HR-NB and, further imply that these miRNAs could play a functional role in tumor progression.
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