Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.

Depletion of ALMS1 inhibits TGF-β/SMAD3/SNAI1 axis altering normal cell migration capacity and epithelial-mesenchyme transition.

Background: ALMS1 is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or epithelial-mesenchymal transition via the TGF-β pathway has not been elucidated yet. Methods: Initially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity. Then, we performed proteomic profiling with TGF-β stimuli in HeLa ALMS1 -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers was validated in hTERT-BJ-5ta. Finally, we also evaluated the SMAD3 phosphorylation in BJ-5ta model and its cell migration capacity. Results: Depletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 -/-. Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction. EMT biomarkers VIM, DSP, and SNAI1 showed an opposite pattern to what would be expected when activating the EMT. Finally, inhibition of SNAI1 was consistent in BJ-5ta model, where a reduced activation of SMAD3 and a decrease in migratory capacity were also observed. Conclusion: ALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 inhibits the signal transduction through the TGF-β/SMAD3/SNAI1, which could be affecting the cell migration capacity and EMT.

First person – Nadine Pollak

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nadine Pollak is first author on ‘ Cell cycle progression and transmitotic apoptosis resistance promote escape from extrinsic apoptosis’, published in JCS. Nadine conducted the research described in this article while a postdoc at the Institute of Cell Biology and Immunology, University of Stuttgart, Germany, initially under the supervision of Prof. Peter Scheurich and subsequently in the lab of Prof. Markus Rehm, where she is now investigating the mechanisms underlying cell fate decisions in response to death stimuli throughout the cell cycle at the single-cell level.

YAP/TAZ: Key Players for Rheumatoid Arthritis Severity by Driving Fibroblast Like Synoviocytes Phenotype and Fibro-Inflammatory Response

ObjectiveThe role of YAP/TAZ, two transcriptional co-activators involved in several cancers, was investigated in rheumatoid arthritis (RA).MethodsFibroblast like synoviocytes (FLS) from patients with RA or osteoarthritis were cultured in 2D or into 3D synovial organoids. Arthritis rat model (n=28) and colitis mouse model (n=21) were used. YAP/TAZ transcriptional activity was inhibited by verteporfin (VP). Multiple techniques were used to assess gene and/or protein expression and/or localization, cell phenotype (invasion, proliferation, apoptosis), bone erosion, and synovial stiffness.ResultsYAP/TAZ were transcriptionally active in arthritis (19-fold increase for CTGF expression, a YAP target gene, in RA vs. OA organoids; p<0.05). Stiff support of culture or pro-inflammatory cytokines further enhanced YAP/TAZ transcriptional activity in RA FLS. Inhibiting YAP/TAZ transcriptional activity with VP restored a common phenotype in RA FLS with a decrease in apoptosis resistance, proliferation, invasion, and inflammatory response. Consequently, VP blunted hyperplasic lining layer formation in RA synovial organoids. In vivo, VP treatment strongly reduced arthritis severity (mean arthritic index at 3.1 in arthritic group vs. 2.0 in VP treated group; p<0.01) by restoring synovial homeostasis and decreasing systemic inflammation. YAP/TAZ transcriptional activity also enhanced synovial membrane stiffening in vivo, thus creating a vicious loop with the maintenance of YAP/TAZ activation over time in FLS. YAP/TAZ inhibition was also effective in another inflammatory model of mouse colitis.ConclusionOur work reveals that YAP/TAZ were critical factors during arthritis. Thus, their transcriptional inhibition could be relevant to treat inflammatory related diseases.

Hippo Pathway-related Genes Expression Is Deregulated in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are hematological disorders characterized by increased proliferation of precursor and mature myeloid cells. MPN patients may present driver mutations in JAK2, MPL, and CALR genes, which are essential to describe the molecular mechanisms of MPN pathogenesis. Despite all the new knowledge on MPN pathogenesis, many questions remain to be answered to develop effective therapies to cure MPN or impair its progression to acute myeloid leukemia. The present study examined the expression levels of the Hippo signaling pathway members in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), as well as the role that they play in disease pathogenesis. The Hippo pathway is a tumor suppressor pathway that participates in the regulation of cell proliferation, differentiation, and death. Our main findings were: (i) expression of tumor suppressor genes from Hippo pathway were downregulated and seemed to be associated with cell resistance to apoptosis and increased proliferation rate; and (ii) Hippo pathway-related gene expression was associated with mutation status in ET and PMF patients. Therefore, the decreased expression of Hippo pathway-related genes may contribute to the malignant phenotype, apoptosis resistance, and cell proliferation in MPN pathogenesis.

The IRF2/CENP-N/AKT signaling axis promotes proliferation, cell cycling and apoptosis resistance in nasopharyngeal carcinoma cells by increasing aerobic glycolysis

Background Centromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown. Methods Abnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT. Results CENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high 18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance. Conclusions The IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.

Effect of Notch1 Signaling on Cellular Proliferation and Apoptosis of Human Laryngeal Carcinoma

The pathological processes of occurrence and development of malignancies include the excessive proliferation and apoptosis resistance of neoplastic cells. The study aims to identify the effects of Notch1 signaling on the proliferation and apoptosis of laryngeal cancer cells in hypoxic microenvironment. Notch1 and Ki-67 expression in laryngeal squamous cell carcinoma (LSCC) tissue samples were detected by immunohistochemistry. The apoptotic index (AI) of LSCC was evaluated by TUNEL method. In laryngeal cancer cells, small interfering RNA (siRNA) technology was to inhibit Notch1 expression. Meanwhile, Real-time PCR detected Notch1, Hes1 and Hey1 mRNA expression, and Western blot detected Notch1 and Notch1 intracellular domain (N1ICD) protein expression. Annexin V-FITC/propidium iodide staining and Cell Counting Kit‑8 methods measured cell apoptosis and proliferation, respectively. Notch1 expression was detected in 63.55％(68/107) of LSCC samples and was significantly related to the proliferation index (PI) (P < 0.05) and AI (P < 0.05) in LSCC tissues. Furthermore, it was confirmed that hypoxia could induce proliferation and inhibit apoptosis of laryngeal carcinoma cells (P < 0.05). Meanwhile, Notch1 expression and Notch1 signaling activity could be upregulated by hypoxia (P < 0.05). In contrast, suppression of Notch1 signaling activity in hypoxic neoplastic cells could obviously decrease cell proliferation and increase cell apoptosis (both P<0.05). Our study has demonstrated that hypoxia may promote cell proliferation and inhibit cell apoptosis of laryngeal carcinoma. Notch1 signalling may exert a pivotal role in regulating the proliferation and apoptosis resistance of laryngeal cancer cells under hypoxia.

Polymorphism rs2682818 participates in the progression of colorectal carcinoma via miR-618-TIMP1 regulatory axis

Colorectal carcinoma (CRC) has a high morbidity and mortality. Current studies have confirmed a variety of microRNA polymorphisms were associated with tumor susceptibility, however, the mechanisms are still unknown. In this study, we were aimed to clarify how polymorphism rs2682818 participated in the progression of CRC. First of all, the differential expression of miR-618 was assessed by quantitative real-time polymerase chain reaction in CRC patients with different genotypes of polymorphism rs2682818, including homozygous (TT) genotype, homozygous (GG) genotype and heterozygous (TG) genotype. Secondly, plasmids carried miR-168 precursor sequences harboring rs2682818 (SNP type) or without rs2682818 (wild type) were transfected into 293T cells to verify that polymorphism rs2682818 affected miR-618 expression. Thirdly, CCK-8 assay, flow cytometry assay, transwell assay and mouse xenograft assay were performed to measure the biological functions of miR-618 in CRC. Fourthly, the candidate target genes of miR-618 which were predicted by bioinformatics tools were verified by luciferase reporter assay. Finally, in order to explain the potential molecular mechanisms, western blotting was performed to demonstrate the differential expression and phosphorylation of pathway related proteins. The results showed that miR-618 was down-regulated in colon cancer, especially in CRC patients with rs2682818 GG homozygous genotype. Higher expression of mature miR-618 occurred in patients with TT homozygous genotype, and these patients usually had a longer survival time. Moreover, miR-618 mimic obviously impaired the growth and invasion ability of CRC cells, and miR-618 mimic also remarkably promoted CRC cell apoptosis. Our luciferase experiments confirmed that TIMP1 was a target of miR-618 in CRC cells. Knockdown of TIMP1 also significantly inhibited the malignant cytological features of CRC, including malignant growth and invasion as well as apoptosis resistance. In summary, polymorphism rs2682818 participated in the progression of CRC via affecting the expression of mature miR-618 in CRC cells, and miR-618 inhibited the progression of CRC via targeting TIMP1expression.