Ancient Adversary - HERV-K (HML-2) in Cancer.

Human endogenous retroviruses (HERV), ancient integrations of exogenous viruses, make up 8% of our genome. Long thought of as mere vestigial genetic elements, evidence is now accumulating to suggest a potential functional role in numerous pathologies including neurodegenerative diseases, autoimmune disorders, and multiple cancers. The youngest member of this group of transposable elements is HERV-K (HML-2). Like the majority of HERV sequences, significant post-insertional mutations have disarmed HERV-K (HML-2), preventing it from producing infectious viral particles. However, some insertions have retained limited coding capacity, and complete open reading frames for all its constituent proteins can be found throughout the genome. For this reason HERV-K (HML-2) has garnered more attention than its peers. The tight epigenetic control thought to suppress expression in healthy tissue is lost during carcinogenesis. Upregulation of HERV-K (HML-2) derived mRNA and protein has been reported in a variety of solid and liquid tumour types, and while causality has yet to be established, progressively more data are emerging to suggest this phenomenon may contribute to tumour growth and metastatic capacity. Herein we discuss its potential utility as a diagnostic tool and therapeutic target in light of the current in vitro, in vivo and clinical evidence linking HERV-K (HML-2) to tumour progression.

Association of GSTM1 and GSTT1 gene polymorphisms with COVID-19 susceptibility and its outcome.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become a global health issue and develops into a broad range of illnesses from asymptomatic to fatal respiratory diseases. SARS-CoV-2 infection is associated with oxidative stress that triggers cytokine production, inflammation, and other pathophysiological processes. Glutathione-S-transferase (GST) is an important enzyme that catalyzes the conjugation of glutathione (GSH) with electrophiles to protect the cell from oxidative damage and participates in the antioxidant defense mechanism in the lungs. Thus, in this study, we investigated the role of GSTM1 and GSTT1 gene polymorphism with COVID-19 susceptibility, as well as its outcome. The study included 269 RT-PCR confirmed COVID-19 patients with mild (n = 149) and severe (n = 120) conditions. All subjects were genotyped for GSTM1 and GSTT1 by multiplex polymerase chain reaction (mPCR) followed by statistical analysis. The frequency of GSTM1-/- , GSTT1-/- and GSTM1-/- /GSTT1-/- was higher in severe COVID-19 patients as compared to mild patients but we did not observe a significant association. In the Cox hazard model, death was significantly 2.28-fold higher in patients with the GSTT1-/- genotype (p = 0.047). In combination, patients having GSTM1+/+ and GSTT1-/- genotypes showed a poor survival rate (p = 0.02). Our results suggested that COVID-19 patients with the GSTT1-/- genotype showed higher mortality.

The Role of Nitric Oxide in Cancer: Master Regulator or NOt?

Nitric oxide (NO) is a key player in both the development and suppression of tumourigenesis depending on the source and concentration of NO. In this review, we discuss the mechanisms by which NO induces DNA damage, influences the DNA damage repair response, and subsequently modulates cell cycle arrest. In some circumstances, NO induces cell cycle arrest and apoptosis protecting against tumourigenesis. NO in other scenarios can cause a delay in cell cycle progression, allowing for aberrant DNA repair that promotes the accumulation of mutations and tumour heterogeneity. Within the tumour microenvironment, low to moderate levels of NO derived from tumour and endothelial cells can activate angiogenesis and epithelial-to-mesenchymal transition, promoting an aggressive phenotype. In contrast, high levels of NO derived from inducible nitric oxide synthase (iNOS) expressing M1 and Th1 polarised macrophages and lymphocytes may exert an anti-tumour effect protecting against cancer. It is important to note that the existing evidence on immunomodulation is mainly based on murine iNOS studies which produce higher fluxes of NO than human iNOS. Finally, we discuss different strategies to target NO related pathways therapeutically. Collectively, we present a picture of NO as a master regulator of cancer development and progression.

RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes.

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.

Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells.

BACKGROUND: MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes. RESULTS: Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs' targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKß, Birc1, Birc2, Bric5, ß-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3ß, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1ß, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs' targets corresponded strongly with poor overall and relapse-free survival. CONCLUSIONS: For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.

Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes.

BACKGROUND: Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure. METHODS: In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes. RESULTS: Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma. CONCLUSION: Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.

Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling.

INTRODUCTION: High-risk neuroblastoma (HR-NB) presenting with hematogenous metastasis is one of the most difficult cancers to cure. Patient survival is poor. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictate tumor progression, relapse/recurrence, and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fit the CSC model. METHODS: Well-characterized clones of metastatic site-derived aggressive cells (MSDACs) from a manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit by examining epithelial-to-mesenchymal transition (EMT) (E-cadherin, N-Cadherin), survival (NFκB P65, p50, IκB and pIκB) and drug resistance (ABCG2) by immunoblotting; pluripotency maintenance (Nanog, SOX2) by immunofluorescence; and EMT and stemness related transcription of 93 genes by QPCR profiling. Plasticity of MSDACs under sequential alternation of culture conditions with serum and serum-free stem-cell conditions was assessed by clonal expansion (BrdU incorporation), tumorosphere formation (anchorage independent growth), EMT and stemness related transcriptome (QPCR profiling) and validated with MYC, SOX2, EGFR, NOTCH1 and CXCL2 immunoblotting. RESULTS: HR-NB MSDACs maintained in alternated culture conditions, serum-free stem cell medium to growth medium with serum and vice versa identified its flexible revocable plasticity characteristics. We observed signatures of stem cell-related molecular responses consistent with phenotypic conversions. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem cell-related signaling events, but also instigated additional events depicting aggressive adaptive plasticity. CONCLUSIONS: Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fit the CSC model, and further identified the intrinsic adaptiveness of the successive phenotype switching that clarifies the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem cell-related molecular rearrangements may hold the key to the switch from favorable disease to HR-NB.

Acquired tumor cell radiation resistance at the treatment site is mediated through radiation-orchestrated intercellular communication.

PURPOSE: Radiation resistance induced in cancer cells that survive after radiation therapy (RT) could be associated with increased radiation protection, limiting the therapeutic benefit of radiation. Herein we investigated the sequential mechanistic molecular orchestration involved in radiation-induced radiation protection in tumor cells. RESULTS: Radiation, both in the low-dose irradiation (LDIR) range (10, 50, or 100 cGy) or at a higher, challenge dose IR (CDIR), 4 Gy, induced dose-dependent and sustained NFκB-DNA binding activity. However, a robust and consistent increase was seen in CDIR-induced NFκB activity, decreased DNA fragmentation, apoptosis, and cytotoxicity and attenuation of CDIR-inhibited clonal expansion when the cells were primed with LDIR prior to challenge dose. Furthermore, NFκB manipulation studies with small interfering RNA (siRNA) silencing or p50/p65 overexpression unveiled the influence of LDIR-activated NFκB in regulating CDIR-induced DNA fragmentation and apoptosis. LDIR significantly increased the transactivation/translation of the radiation-responsive factors tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), cMYC, and SOD2. Coculture experiments exhibit LDIR-influenced radiation protection and increases in cellular expression, secretion, and activation of radiation-responsive molecules in bystander cells. Individual gene-silencing approach with siRNAs coupled with coculture studies showed the influence of LDIR-modulated TNF-α, IL-1α, cMYC, and SOD2 in induced radiation protection in bystander cells. NFκB inhibition/overexpression studies coupled with coculture experiments demonstrated that TNF-α, IL-1α, cMYC, and SOD2 are selectively regulated by LDIR-induced NFκB. CONCLUSIONS: Together, these data strongly suggest that scattered LDIR-induced NFκB-dependent TNF-α, IL-1α, cMYC, and SOD2 mediate radiation protection to the subsequent challenge dose in tumor cells.