Abstract Many types of human cells self-destruct to maintain biological homeostasis and defend the body against pathogenic substances. This process, called regulated cell death (RCD), is important for various biological activities, including the clearance of aberrant cells. Thus, RCD pathways represented by apoptosis have increased in importance as a target for the development of cancer medications in recent years. However, because tumor cells show avoidance to apoptosis, which causes treatment resistance and recurrence, numerous studies have been devoted to alternative cancer cell mortality processes, namely necroptosis, pyroptosis, ferroptosis, and cuproptosis; these RCD modalities have been extensively studied and shown to be crucial to cancer therapy effectiveness. Furthermore, evidence suggests that tumor cells undergoing regulated death may alter the immunogenicity of the tumor microenvironment (TME) to some extent, rendering it more suitable for inhibiting cancer progression and metastasis. In addition, other types of cells and components in the TME undergo the abovementioned forms of death and induce immune attacks on tumor cells, resulting in enhanced antitumor responses. Hence, this review discusses the molecular processes and features of necroptosis, pyroptosis, ferroptosis, and cuproptosis and the effects of these novel RCD modalities on tumor cell proliferation and cancer metastasis. Importantly, it introduces the complex effects of novel forms of tumor cell death on the TME and the regulated death of other cells in the TME that affect tumor biology. It also summarizes the potential agents and nanoparticles that induce or inhibit novel RCD pathways and their therapeutic effects on cancer based on evidence from in vivo and in vitro studies and reports clinical trials in which RCD inducers have been evaluated as treatments for cancer patients. Lastly, we also summarized the impact of modulating the RCD processes on cancer drug resistance and the advantages of adding RCD modulators to cancer treatment over conventional treatments. 

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Targeting cell death pathways for cancer therapy: recent developments in necroptosis, pyroptosis, ferroptosis, and cuproptosis research

A decreased physical fitness has been reported in patients and survivors of acute lymphoblastic leukemia (ALL). This is influenced by the negative effects of the disease and by the treatments of childhood cancer. In the past, children were advised to recover in bed, and to take as much relax as possible. Nowadays, it is considered that too much immobility may result in a further decrease of physical fitness and functioning. Exercise training for ALL children has frequently been reported to improve physical fitness and the well-being of the children, since it prevents the negative effects of a sedentary life-style, such as obesity and a poor skeletal health. In recent years, different studies and protocols on this subject has become available for children and young adults with cancer, both during and after treatment. The efficacy of recent physical exercise training interventions, that act on several ALL impairments in children such as skeletal, musculoskeletal, neuromuscular, cardiopulmonary and cardiovascular systems, fatigue, body balance disorders and metabolism alterations have been examined. These side effects might be prevented or significantly reduced by introducing a physical exercise program during or shortly after cancer treatment. Several interventions are discussed and presented for each impairment, reducing their level caused by the disease and thus suggesting the importance of physical training activity in ameliorating the children quality of life.

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Physical training interventions for children and teenagers affected by acute lymphoblastic leukemia and related treatment impairments.

Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2 mM) for 24 h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.

NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells.

Objective Acute lymphoblastic leukemia (ALL) is a frequent malignancy in childhood. The present study was aimed to investigate the effect of miR-223 in ALL and its underlying molecular mechanisms. Methods The mRNA expression of miR-223 and FOXO1 was detected by qRT-RCR in ALL children. The correlation between miR-223 and clinical indexes of ALL was determined. CCRF-CEM and NALM-6 cells were transfected with miR-223 mimic and miR-223 inhibitor, respectively. The proliferation, apoptosis, invasion and migration of CCRF-CEM and NALM-6 cells were measured by MTT, flow cytometry and transwell assay. The protein expression of FOXO1 was detected by Western blot. Additionally, dual-luciferase reporter and RNA pull-down assay were performed to investigate the target gene of miR-223 and validate their targeting relationship. Results The mRNA expression of miR-223 was markedly down-regulated in ALL, but FOXO1 was up-regulated. The protein expression of FOXO1 was highly expressed in CCRF-CEM and NALM-6 cells. The expression of miR-223 was related to WBC, PLT, RBC and risk stratification. Overexpression of miR-223 not only inhibited cell proliferation, migration and invasion, but also induced cell apoptosis. Importantly, FOXO1 was a target gene of miR-223 in ALL cells. Silencing of FOXO1 reversed the effects of miR-223 inhibitor on cell proliferation, migration, invasion and apoptosis in ALL. Conclusions miR-223 could inhibit cell proliferation, migration and invasion, and promote apoptosis by targeting FOXO1 in ALL.

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MicroRNA-223 decreases cell proliferation, migration, invasion, and enhances cell apoptosis in childhood acute lymphoblastic leukemia via targeting Forkhead box O 1.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.

https://www.mdpi.com/1422-0067/22/5/2771/pdf

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia.

Background Necroptosis, is intimately linked to tumor development and prognosis and has been considered as a target for anticancer therapy. However, the role of necroptosis-related genes (NRGs) in colon cancer is unclear. Methods In the present study, we screened 76 NRGs from previous studies and described the landscape of transcriptomic and genetic variation of NRGs in colon cancer (CC) patient samples. Molecular subtypes of necroptosis in colon cancer were identified by clustering analysis, and these molecular subtypes were linked to patient prognosis and TME cell infiltration characteristics. Then, the NRS-score for predicting overall survival (OS) was built based on the TCGA database and validated in the GSE39582 cohort for its predictive power in CC patients. Besides, the ESTIMATE and CIBERSORT algorithms were applied to explore the relationship between NRS-score and tumor immune microenvironment. Results We identified two molecular subtypes associated with necroptosis in CC, which have diverse prognosis and immune microenvironment characteristics. Based on the differentially expressed genes between the two molecular subtypes, we further developed a necroptosis risk score signature, referred to as NRS-score. High NRS-score was associated with poor prognosis in CC through immunosuppressive microenvironment and immune escape mechanisms. The nomogram based on NRS-score showed excellent ability to predict prognosis. In addition, NRS-score presented a positive correlation with tumor mutational burden (TMB) and immune checkpoint blockade (ICB) expression and was closely correlated with multiple anticancer agent susceptibility. Conclusion This work revealed a close relationship between necroptosis and the prognosis and immune microenvironment of colon cancer. The NRS-score based on the 8-gene signature may be used to predict the sensitivity of immunotherapy and chemotherapy in colon cancer patients, and provides a foundation for future studies targeting necroptosis and its immune microenvironment.

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Integrated Analysis of Necroptosis-Related Genes for Prognosis, Immune Microenvironment Infiltration, and Drug Sensitivity in Colon Cancer

Ikaros isoform 6 (Ik6) is associated with a poor prognosis for children with acute lymphoblastic leukemia (ALL). Our previous study demonstrated that overexpression of Ik6 enhances proliferation and chemoresistance of leukemia cells, with a possible underlying mechanism that involves antiapoptosis. In the present study, we investigated whether Ik6 protects against apoptosis by regulating the Akt-FoxO1 pathway. Bone marrow samples from children with ALL were collected and evaluated. In Ik6+ patients, the Akt-FoxO1 pathway was activated such that expression of phosphorylated Akt and FoxO1 was significantly increased, but that of Bim and p27 decreased. In vitro experiments in this study were performed by using human ALL Nalm-6 cells that were stably transfected with Ik6 (Nalm-6/Ik6) or Sup-B15 and Ik6 shRNA (Sup-B15/Ik6 shRNA). Upon treatment with daunorubicin, Nalm-6/Ik6 cells exhibited a statistically significant reduction in apoptosis, with increased expression of p-Akt and p-FoxO1. In contrast, an increase in apoptosis with decreased expression of p-Akt and p-FoxO1 was observed in Sup-B15/Ik6 shRNA cells. This protection was dependent on activation of caspase-3 cleavage. By using an activator and an inhibitor of Akt or FoxO1, we demonstrated that Akt or FoxO1 activation had no effect on Ik6 expression. In conclusion, Ik6, the upstream factor of Akt-FoxO1 pathway, can protect ALL cells against daunorubicin-induced apoptosis and can potentially be explored as a therapeutic target in the treatment of patients with ALL.

https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.2A0116-040RR

Ikaros 6 protects acute lymphoblastic leukemia cells against daunorubicin‐induced apoptosis by activating the Akt‐FoxO1 pathway

// Fen Zhou 1,2 , Meiling Zhang 1,2 , Juan Han 1,2 , Jinjin Hao 1,2 , Yan Xiao 1,2 , Qin Liu 1,2 , Runming Jin 1,2 , Heng Mei 2 1 Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 2 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Correspondence to: Runming Jin, email: // Heng Mei, email: // Keywords : skeletal abnormalities, SPECT, acute lymphoblastic leukemia, relapse Received : July 26, 2016 Accepted : May 08, 2017 Published : May 23, 2017 Abstract Objectives: Most children with acute lymphoblastic leukemia (ALL) exhibit skeletal abnormalities. This study aimed to investigate bone lesions detected by whole-body bone single-photon emission computed tomography (SPECT) and its prognostic value in children with ALL. Methods: A retrospective analysis was performed using whole-body bone SPECT scans obtained from children with ALL in our department between June 2008 and June 2012. A total of 166 children newly diagnosed with ALL were included, and the patients were divided into two groups: patients with positive and negative SPECT scans. We compared the clinical characteristics of the two groups and analyzed the relationship between the skeletal abnormalities detected by SPECT and prognosis. Results: Among the 166 patients, bone scintigraphic abnormalities was detected by SPECT scan in sixty-four patients (38.6%). The most common site was the limbs. There were no significant differences in age, gender, WBC count at diagnosis, risk group and minimal residual disease (MRD) level between SPECT-positive patients and their SPECT-negative counterparts. The event-free and overall survival rates were higher in SPECT-positive patients, but the difference was not statistically significant. However, patients with positive SPECT scans, especially those with multifocal abnormalities (≥3 sites), had a higher rate of relapse ( P < 0.05). Multivariate analyses identified that abnormal SPECT scan (HR = 3.547, P = 0.015) was an independent relapse risk. Conclusion: Children with ALL and multiple skeletal abnormalities will suffer from relapse. Abnormal SPECT scan was associated with increased relapse risk which might be a potential relapse marker for ALL children.

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Meiling Zhang

Papers

Integrated Analysis of Necroptosis-Related Genes for Prognosis, Immune Microenvironment Infiltration, and Drug Sensitivity in Colon Cancer

Ikaros 6 protects acute lymphoblastic leukemia cells against daunorubicin‐induced apoptosis by activating the Akt‐FoxO1 pathway

Skeletal abnormalities detected by SPECT is associated with increased relapse risk in pediatric acute lymphoblastic leukemia.