Three-dimensional genome landscape comprehensively reveals patterns of spatial gene regulation in papillary and anaplastic thyroid cancers: a study using representative cell lines for each cancer type.

BACKGROUND: Spatial chromatin structure is intricately linked with somatic aberrations, and somatic mutations of various cancer-related genes, termed co-mutations (CoMuts), occur in certain patterns during cancer initiation and progression. The functional mechanisms underlying these genetic events remain largely unclear in thyroid cancer (TC). With discrepant differentiation, papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) differ greatly in characteristics and prognosis. We aimed to reveal the spatial gene alterations and regulations between the two TC subtypes. METHODS: We systematically investigated and compared the spatial co-mutations between ATC (8305C), PTC (BCPAP and TPC-1), and normal thyroid cells (Nthy-ori-3-1). We constructed a framework integrating whole-genome sequencing (WGS), high-throughput chromosome conformation capture (Hi-C), and transcriptome sequencing, to systematically detect the associations between the somatic co-mutations of cancer-related genes, structural variations (SVs), copy number variations (CNVs), and high-order chromatin conformation. RESULTS: Spatial co-mutation hotspots were enriched around topologically associating domains (TADs) in TC. A common set of 227 boundaries were identified in both ATC and PTC, with significant overlaps between them. The spatial proximities of the co-mutated gene pairs in the two TC types were significantly greater than in the gene-level and overall backgrounds, and ATC cells had higher TAD contact frequency with CoMuts > 10 compared with PTC cells. Compared with normal thyroid cells, in ATC the number of the created novel three-dimensional chromatin structural domains increased by 10%, and the number of shifted TADs decreased by 7%. We found five TAD blocks with CoMut genes/events specific to ATC with certain mutations in genes including MAST-NSUN4, AM129B/TRUB2, COL5A1/PPP1R26, PPP1R26/GPSM1/CCDC183, and PRAC2/DLX4. For the majority of ATC and PTC cells, the HOXA10 and HIF2α signals close to the transcription start sites of CoMut genes within TADs were significantly stronger than those at the background. CNV breakpoints significantly overlapped with TAD boundaries in both TC subtypes. ATCs had more CNV losses overlapping with TAD boundaries, and noncoding SVs involved in intrachromosomal SVs, amplified inversions, and tandem duplication differed between ATC and PTC. TADs with short range were more abundant in ATC than PTC. More switches of A/B compartment types existed in ATC cells compared with PTC. Gene expression was significantly synchronized, and orchestrated by complex epigenetics and regulatory elements. CONCLUSION: Chromatin interactions and gene alterations and regulations are largely heterogeneous in TC. CNVs and complex SVs may function in the TC genome by interplaying with TADs, and are largely different between ATC and PTC. Complexity of TC genomes, which are highly organized by 3D genome-wide interactions mediating mutational and structural variations and gene activation, may have been largely underappreciated. Our comprehensive analysis may provide key evidence and targets for more customized diagnosis and treatment of TC.

GELAD chemotherapy with sandwiched radiotherapy for patients with newly diagnosed stage IE/IIE natural killer/T-cell lymphoma: a prospective multicentre study.

Early-stage natural killer/T-cell lymphoma (NK/TCL) patients usually receive a combination of chemotherapy and radiotherapy, but the optimal treatment approach has not yet been established. This study aimed to investigate the efficacy and safety profile of a novel chemotherapy regimen and sandwiched radiotherapy in early-stage NK/TCL. Patients with newly diagnosed stage IE/IIE disease were eligible. Patients were initially treated with two courses of the GELAD regimen (gemcitabine 1·0 g/m2 day 1, etoposide 60 mg/m2 days 1-3, pegaspargase 2000 units/m2 day 4, and dexamethasone 40 mg days 1-4), followed by intensity-modulated radiotherapy (IMRT; 50-56 Gy in 25-28 fractions) and two additional courses of GELAD chemotherapy. A total of 52 patients were enrolled. The overall response rate and complete response rate per Lugano 2014 criteria were 94·2% and 92·3% respectively. With a median follow-up of 32 months, the estimated four-year overall survival rate and progression-free survival rate were 94·2% [95% confidence interval (CI), 83·2% to 93·1%] and 90·4% (95% CI, 78·4% to 95·9%) respectively. The most common adverse events were related to pegaspargase. Haematological toxicities were mild, with grade 3/4 neutropenia in 15·4% of patients. Our study provides a new approach with high activity and improved safety for the treatment of early-stage NK/TCL patients. This study was registered at www.clinicaltrials.gov as NCT02733458.

Label-Free Electrochemical Sensor for CD44 by Ligand-Protein Interaction.

Detection of biomarkers in biosystems plays a key role in advanced biodiagnostics for research and clinical use. Design of new analytical platforms is challenging and in demand, addressing molecular capture and subsequent quantitation. Herein, we developed a label-free electrochemical sensor for CD44 by ligand-protein interaction. We assembled carbon nanotube composites on the electrode to enhance electronic conductivity by 6.2-fold and reduce overpotential with a shift of 77 mV. We conjugated hyaluronic acid (HA) to the surface of carbon nanotubes via electrostatic interaction between HA and poly(diallyldimethylammonium chloride) (PDDA). Consequently, we performed direct electrochemical sensing of CD44 with a dynamic range of 0.01-100 ng/mL and detection limit of 5.94 pg/mL without any postlabeling for amplification, comparable to the best current results. The sensor also displayed high selectivity, reproducibility with relative standard deviation (RSD, n = 5) of 2.57%, and long-term stability for 14 days. We demonstrated applications of the sensor in detection of human serum and cancer cells. Our work guides the development of more sensor types by ligand-protein interactions and contributes to design of interfaces in given biosystems for diagnosis.

Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

Rhabdomyosarcoma (RMS) represents one of the most lethal soft-tissue sarcomas in children. The toxic trace element arsenic has been reported to function as a radiosensitizer in sarcomas. To investigate the role of arsenic sulfide (As4S4) in enhancing radiation sensitization in RMS, this study was conducted to elucidate its underlying mechanism in radiotherapy. The combination of As4S4 and radiotherapy showed significant inhibition in RMS cells, as demonstrated by the cell counting kit-8 (CCK-8) assay and flow cytometry. Subsequently, we demonstrated for the first time that As4S4, as well as the knockdown of NFATc3 led to double-strand break (DSB) through increased expression of RAG1. In vivo experiment confirmed that co-treatment efficiently inhibited RMS growth. Furthermore, survival analysis of a clinical cohort consisting of 59 patients revealed a correlation between NFATc3 and RAG1 expression and overall survival (OS). Cox regression analysis also confirmed the independent prognostic significance of NFATc3 and RAG1.Taken together, As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB. NFATc3 and RAG1 are potential therapeutic targets. As4S4 will hopefully serve as a prospective radio-sensitizing agent for RMS.

Relationship Between Autophagy and Drug Resistance in Tumors.

Multidrug Resistance (MDR) in tumor cells, a phenomenon in which tumor cells become resistant to chemotherapeutic drugs with different chemical structures and mechanisms of action, is a major obstacle to tumor therapy and is an urgent problem to be addressed. Autophagy, widely found in eukaryotic cells, is a lysosome-dependent pathway of self-degradation. In different environments, autophagy can play different roles in the self-protection of cells. At different stages of tumorigenesis, autophagy can play two distinct roles: inhibition of cancer and promotion of cancer. The relationship between autophagy and drug resistance in tumor cells is complex. Moreover, autophagy can play a role in promoting drug resistance and drug sensitivity through different molecular pathways. This study aimed to investigate the relationship between autophagy and drug resistance in tumor cells from the perspective of molecular mechanisms.

Arsenic Sulfide Inhibits Hepatocellular Carcinoma Metastasis by Suppressing the HIF-1α/VEGF Pathway.

BACKGROUND: Metastasis is one of the principal reasons of cancer mortality from hepatocellular carcinoma (HCC). The goal of our investigation was to examine the mechanism by which arsenic sulfide (As4S4) represses the metastasis of HCC. METHODS: The cell counting kit-8 (CCK-8) assay was conducted to observe cell viability of HCC cell lines HepG2 and Hep3B following As4S4 treatment, and their metastasis was studied using the wound-healing and transwell assays. HCC-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) was assessed by tube formation assay. Enzyme-linked immunosorbent assay (ELISA), western blot, quantitative polymerase chain reaction and immunofluorescence staining were utilized to evaluate key molecules involved in metastasis, including hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), Vimentin, N-cadherin and E-cadherin. RESULTS: As4S4 suppressed the proliferation, migration and invasion of HepG2 and Hep3B cell lines in a concentration-dependent pattern, and inhibited HCC cell-induced angiogenesis of HUVEC in the tube formation assay. Treatment with As4S4 also decreased the expression of crucial elements involved in the metastasis of HCC cells, including HIF-1α and VEGF, while reducing epithelial-mesenchymal transition, as shown by Western blot, ELISA and immunofluorescence staining. CONCLUSIONS: Overall, results above indicate that As4S4 suppresses the metastasis of HCC cells via the HIF-1α/VEGF pathway.

RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein.

Cancer stem cells (CSCs) contribute to radioresistance in medulloblastoma. Thus, identification of key regulators of medulloblastoma stemness is critical for improving radiotherapy for medulloblastoma. In the present study, we profiled CSC-related long non-coding RNAs (lncRNAs) between radioresistant and parental medulloblastoma cells. The roles of the lncRNA RBM5-AS1 in the stemness and radiosensitivity of medulloblastoma cells were investigated. We found that RBM5-AS1, a novel inducer of medulloblastoma stemness, was significantly upregulated in radioresistant medulloblastoma cells compared to parental cells. Knockdown of RBM5-AS1 diminished the viability and clonogenic survival of both radioresistant and parental medulloblastoma cells after radiation. Silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis and DNA damage. In vivo studies confirmed that depletion of RBM5-AS1 inhibited tumor growth and increased radiosensitivity in a medulloblastoma xenograft model. In contrast, overexpression of RBM5-AS1 reduced radiation-induced apoptosis and DNA damage in medulloblastoma cells. Mechanistically, RBM5-AS1 interacted with and stabilized sirtuin 6 (SIRT6) protein. Silencing of SIRT6 reduced the stemness and reinforced radiation-induced DNA damage in medulloblastoma cells. Overexpression of SIRT6 rescued medulloblastoma cells from RBM5-AS1 depletion-induced radiosensitization and DNA damage. Overall, we identify RBM5-AS1 as an inducer of stemness and radioresistance in medulloblastoma. Targeting RBM5-AS1 may represent a potential strategy to overcome the resistance to radiotherapy in this malignancy.

Arsenic sulfide induces miR-4665-3p to inhibit gastric cancer cell invasion and migration.

PURPOSE: Gastric carcinogenesis is a multistep process and is the second-highest cause of cancer death worldwide with a high incidence of invasion and metastasis. MicroRNAs (miRNAs) engage in complex interactions with the machinery that controls the transcriptome and concurrently target multiple mRNAs. Recent evidence has shown that miRNAs are involved in the cancer progression, including promoting cell-cycle, conferring resistance to apoptosis, and enhancing invasiveness and metastasis. Here, we aim to elucidate the roles of miRNAs, especially microRNA-4665-3p (miR-4665-3p), in the inhibitory effect of arsenic sulfide in gastric cancer (GC). METHODS: The arsenic sulfide-induced miRNA expression alterations in AGS cells was determined by miRNA microarray. RT-PCR was used to further verify the arsenic sulfide-regulated miRNAs in GC tissues. The inhibition of miR-4665-3p on the migration and invasion of GC cells were determined by wound healing assay and transwell assay. Western blot analysis was used to detect the expression of EMT related proteins and the putative target of miR-4665-3p. RESULTS: The miR-4665-3p was up-regulated by arsenic sulfide and showed inhibition upon the migration and invasion of GC cells. MiRBase and Western blotting indicated that miR-4665-3p directly down-regulated the oncoprotein GSE1. Morphological observation also indicated that the up-regulation of miR-4665-3p inhibits the EMT in GC cells. CONCLUSION: Our data demonstrates that the increased expression of miR-4665-3p induced by arsenic sulfide suppresses the cell invasion, metastasis and EMT of GC cells, and has the potential to be a novel therapeutic target in GC.

Scattered Dose to Ovary From Radiotherapy for Neuroblastoma in Female Children.

PURPOSE: To measure the scattered dose to ovary from radiotherapy for neuroblastoma in female children and to evaluate the relevant risks for radiation-induced ovarian damage. MATERIAL AND METHODS: Radiotherapy for child neuroblastoma was simulated on the water phantom. The scattered dose to ovary is measured by ionization chamber on the linear accelerator with 3-dimensional conformal radiation therapy and intensity-modulated radiation therapy treatment producing 6MV and 10MV X-rays. The treatment planning procedure was carried out on a computer system (TPS, Oncentra). Optimization of the number and orientation of beams were performed in order to minimize the ovarian dose. RESULTS: For the target dose of 21.6 Gy, the scattered dose to ovary was ranged from 1.3 to 46.8 cGy depending on the treatment method and the energy of the beams. The ovarian dose of intensity-modulated radiation therapy is 1.32 to 1.64 times higher than that of 3-dimensional conformal radiation therapy. The ovarian dose of 6MV beam's energy is 1.52 to 1.64 times higher than that of 10MV beam's energy. For the radiotherapy, the scattered dose of ovaries on phantom by ionization chamber was 1.40 to 2.32 times higher than that on TPS calculated. CONCLUSION: The dosimetric data suggest that pediatric radiotherapy is not associated with a risk for permanent damage to the ovaries in female children. Through choosing the beams' energy and treatment plan's method, the scattered dose of ovaries can be reduced. The risk for development of hereditary disorders in offspring conceived after exposure is low.

Arsenic sulfide induces RAG1-dependent DNA damage for cell killing by inhibiting NFATc3 in gastric cancer cells.

BACKGROUND: Arsenic sulfide was found to have potential anti-cancer activities, especially in gastric cancer. However, the underlying mechanism need to be further explored. This study was aimed to investigate the mechanism of arsenic compounds on gastric cancer. METHODS: Gastric cancer cell lines were infected with lentiviral vector carrying shNFATc3 and/or treated with arsenic sulfide. MTT assay were performed to assess cell growth. Flow cytometer assays were used to detect cell cycle and reactive oxygen species (ROS) level of gastric cancer cells. Western blot was carried out to detect nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3), cell cycle markers, DNA damage pathway protein expression as well as other protein expression in gastric cancer cell lines. The expression of recombination activating gene 1 (RAG1) in gastric cancer cell lines was determined by RNA-sequencing analyses and Real-Time qPCR. The effect of NFATc3 on RAG1 were determined by CHIP-qPCR assay. The effect of arsenic sulfide on AGS cells was evaluated in vivo. RESULTS: We show that arsenic sulfide as well as knockdown of NFATc3 resulted in increased double-strand DNA damage in gastric cancer cells by increasing the expression of RAG1, an endonuclease essential for immunoglobulin V(D) J recombination. Overexpression of NFATc3 blocked the expression of RAG1 expression and DNA damage induced by arsenic sulfide. Arsenic sulfide induced cellular oxidative stress to redistribute NFATc3, thereby inhibiting its transcriptional function, which can be reversed by N-acetyl-L-cysteine (NAC). We show that NFATc3 targets the promoter of RAG1 for transcriptional inhibition. We further showed that NFATc3 upregulation and RAG1 downregulation significantly associated with poor prognosis in patients with gastric cancer. Our in vivo experiments further confirmed that arsenic sulfide exerted cytotoxic activity against gastric cancer cells through inhibiting NFATc3 to activate RAG1 pathway. CONCLUSION: These results demonstrate that arsenic sulfide targets NFATc3 to induce double strand DNA break (DSB) for cell killing through activating RAG1 expression. Our results link arsenic compound to the regulation of DNA damage control and RAG1 expression as a mechanism for its cytotoxic effect.

[Finite element analysis on the effect of lateral wedge insole intervention on the contact characteristics of the subtalar joint].

OBJECTIVE: To establish a three-dimensional finite element model of the lower limb bones, and investigate the changes of the contact characteristics of the subtalar joint after using laterally wedge insole intervention. METHODS: Using the reverse modeling technology, the lower limb bones of normal adult volunteers was scanned by CT. Mimics 10.0 and Geomagic Studio 6.0 software were used to reconstruct the 3D morphology of bones and external soft tissue of the feet. The laterally wedge insole was designed in ProE 5.0. And then all the models were imported into Hyperwork 10.0 and meshed, and given the material properties. The finite element analysis was carried out in ABAQUS 6.9. RESULTS: A three-dimensional finite element model of the lower extremity was established, which was consisted of 95 365 nodes and 246 238 elements. The contact area of the standing state of the lower joint was larger than that of the anterior middle joint surface. The peak stress was concentrated in the anterior lateral part of the posterior articular surface, and the average stress value was(3.85±1.03) MPa. Compared with the model of 0°, the contact area of the subtalar joint was reduced accordingly. There was a significant correlation between anterior middle joint |r|=0.964, P=0.008, and posterior articular |r|=0.978, P=0.002. The equivalent stress of 0° model distributed from(3.07±1.14) MPa to(3.85± 1.03) MPa, which had no statistically difference. Compared with the 0° model, the equivalent stress of the anterior and middle joint surfaces of the 8° model was significantly reduced(P<0.05), but the peak stress of the posterior articular surface was significantly increased(P<0.05). In the 12° model, the peak stress was sharply increased to(10.51±3.53) MPa. Compared with 8° model, there was no statistically difference(P<0.05). Although the peak stress was slightly increased in 16° model, but compared with 12° model, there was no statistically differences(P>0.05). CONCLUSIONS: Although a certain valgus can be obtained in subtalar by wearing LWI, the result comes at the cost of the stress concentration on posterior surface. Through this study, we can find that LWI with 8° tilt angle could provide appropriate valgus moment without causing excessive concentration. Therefore, in order to avoid secondary ankle complications, we should not increase the tilt angle blindly.

microRNA-539 suppresses tumor growth and tumorigenesis and overcomes arsenic trioxide resistance in hepatocellular carcinoma.

AIMS: Dysregulation of microRNAs (miRNAs) plays a critical role in tumor growth and progression. In this study, we sought to explore the expression and biological roles of miR-539 in hepatocellular carcinoma (HCC). MAIN METHODS: The expression of miR-539 in human HCC tissues and cell lines was examined. The effects of miR-539 overexpression on cell growth, tumorigenicity, arsenic trioxide resistance of HCC cells were determined. The signaling pathways involved in the action of miR-539 in HCC were also investigated. KEY FINDINGS: miR-539 was downregulated in HCC tissues and cells, relative to corresponding controls. Overexpression of miR-539 inhibited HCC cell viability and colony formation in vitro and impaired tumorigenesis of HCC cells in vivo. Transfection with miR-539 mimic significantly induced apoptosis in HepG2 cells, which was coupled with reduced expression of anti-apoptotic proteins Bcl-2 and Bcl-xL and decreased phosphorylation of Stat3. Overexpression of a constitutively active form of Stat3 partially blocked miR-539-mediated apoptosis. Enforced expression of miR-539 resensitized arsenic trioxide-resistant HCC cells to arsenic trioxide. Intratumoral delivery of miR-539 mimic significantly retarded the growth of xenograft tumors from arsenic trioxide-resistant HCC cells by about 35%, compared to delivery of control miRNA (P<0.05). In combination with arsenic trioxide, miR-539 mimic yielded about 80% decrease in tumor burden. SIGNIFICANCE: miR-539 functions as a tumor suppressor in HCC and reexpression of this miRNA offers a potential therapeutic strategy for this disease.

Simultaneous integrated boost in breast conserving radiotherapy: is replanning necessary following tumor bed change?

Tumor bed (TB) change is often seen during the whole breast irradiation (WBI) in early breast cancer patients. The aims of this study were to evaluate change in seroma volume on repeat CTs and to explore whether replanning is necessary in breast conserving radiotherapy (RT) using the intensity modulated radiotherapy (IMRT) with simultaneous integrated boost (SIB). Thirty patients underwent WBI with 84 CT scans (24 with 3 CTs (CT1, CT2 and CT3) and 6 without CT3) during the 6 weeks of RT were reviewed. TB and other target volumes on all CTs were delineated and compared. IMRT-SIB treatment plans with 50.68 Gy to the whole breast and 64.4 Gy to the boost in 28 fractions were constructed in the CT1. Replan and hybrid plan (without replanning) in the CT2 were reproduced. Dosimetric differences between the replans and hybrid plans were also compared. The mean TB volumes for CT1, CT2 and CT3 were 42.1 cm(3), 20.1 cm(3), 17.0 cm(3), respectively. The mean TB volume reduction was 40.5% from the CT1 to CT2 and 4.3% from the CT2 to CT3. The difference of TB volumes between the CT1 and CT2 was statistically significance (p < 0.001), but not significant between the CT2 and CT3 (p = 1.000). For all patients, target coverage remained adequate with either hybrid plans or replans. However, replanning can significantly decrease the whole breast mean dose (35.2 vs. 35.6 Gy, p = 0.026) and breast volume outside the boost receiving 95% of the boost prescribed dose (39.5 vs. 68.2 cm(3), p < 0.001). In summary, boost volume could irradiate adequately after the significance shrinkage of tumor bed with the fractionated schedule of IMRT-SIB, but replanning on a second CT could avoid the undesired high dose irradiated breast tissue outside boost.

Post mastectomy linac IMRT irradiation of chest wall and regional nodes: dosimetry data and acute toxicities.

BACKGROUND: Conventional post-mastectomy radiation therapy is delivered with tangential fields for chest wall and separate fields for regional nodes. Although chest wall and regional nodes delineation has been discussed with RTOG contouring atlas, CT-based planning to treat chest wall and regional nodes as a whole target has not been widely accepted. We herein discuss the dosimetric characteristics of a linac IMRT technique for treating chest wall and regional nodes as a whole PTV after modified radical mastectomy, and observe acute toxicities following irradiation. METHODS: Patients indicated for PMRT were eligible. Chest wall and supra/infraclavicular region +/-internal mammary nodes were contoured as a whole PTV on planning CT. A simplified linac IMRT plan was designed using either integrated full beams or two segments of half beams split at caudal edge of clavicle head. DVHs were used to evaluate plans. The acute toxicities were followed up regularly. RESULTS: Totally, 85 patients were enrolled. Of these, 45 had left-sided lesions, and 35 received IMN irradiation. Planning designs yielded 55 integrated and 30 segmented plans, with median number of beams of 8 (6-12). The integrated and segmented plans had similar conformity (1.41±0.14 vs. 1.47±0.15, p=0.053) and homogeneity indexes (0.13±0.01 vs. 0.14±0.02, p=0.069). The percent volume of PTV receiving >110% prescription dose was <5%. As compared to segmented plans, integrated plans typically increased V5 of ipsilateral lung (p=0.005), and heart (p=0.001) in patients with left-sided lesions. Similarly, integrated plans had higher spinal cord Dmax (p=0.009), ipsilateral humeral head (p<0.001), and contralateral lung Dmean (p=0.019). During follow-up, 36 (42%) were identified to have ≥ grade 2 radiation dermatitis (RD). Of these, 35 developed moist desquamation. The median time to onset of moist desquamation was 6 (4-7) weeks from start of RT. The sites of moist desquamation were most frequently occurred in anterior axillary fold (32/35), and secondly chest wall (12/35). The difference in occurrence of ≥ grade 2 RD between integrated and segmented plans was statistically insignificant (X2=0.35, p=0.55). Only 2 were found to have grade 2 radiation pneumonitis. CONCLUSIONS: The linac IMRT technique applied in PMRT with chest wall and regional nodes as a whole PTV was dosimetrically feasible, and the treatment was proved to be well-tolerated by most patients.