A highly sensitive nanopore platform for measuring RNase A activity.

Ribonuclease A (RNase A) plays significant roles in several physiological and pathological conditions and can be used as a valuable diagnostic biomarker for human diseases such as myocardial infarction and cancer. Hence, it is of great importance to develop a rapid and cost-effective method for the highly sensitive detection of RNase A. The significance of RNase A assay is further enhanced by the growing attention from the biotechnology and pharmaceutical industries to develop RNA-based vaccines and drugs in large part as a result of the successful development of mRNA vaccines in the COVID-19 pandemic. Herein, we report a label-free method for the detection of RNase A by monitoring its proteolytic cleavage of an RNA substrate in a nanopore. The method is ultra-sensitive with the limit of detection reaching as low as 30 fg per milliliter. Furthermore, sensor selectivity and the effects of temperature, incubation time, metal ion, salt concentration on sensor sensitivity were also investigated.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability.

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis.

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

Handling Incomplete or Late-Onset Toxicities in Early-Phase Dose-Finding Clinical Trials: Current Practice and Future Prospects.

PURPOSE: The way late-onset toxicities are managed can affect trial outcomes and participant safety. Specifically, participants often might not have completed their entire follow-up period to observe any toxicities before new participants would be recruited. We conducted a methodological review of published early-phase dose-finding clinical trials that used designs accounting for partial and complete toxicity information, aiming to understand (1) how such designs were implemented and reported and (2) if sufficient information was provided to enable the replicability of trial results. METHODS: Until March 26, 2023, we identified 141 trials using the rolling 6 design, the time-to-event continuous reassessment method (TITE-CRM), the TITE-CRM with cycle information, the TITE Bayesian optimal interval design, the TITE cumulative cohort design, and the rapid enrollment design. Clinical settings, design parameters, practical considerations, and dose-limiting toxicity (DLT) information were extracted from these published trials. RESULTS: The TITE-CRM (61, 43.3%) and the rolling 6 design (76, 53.9%) were most frequently implemented in practice. Trials using the TITE-CRM had longer DLT assessment windows beyond the first cycle compared with the rolling 6 design (52.5% v 6.6%). Most trials implementing the TITE-CRM (91.8%, 56 of 61) failed to describe essential parameters in the protocols or the study result papers. Only five TITE-CRM trials (8.2%, 5 of 61) reported sufficient information to enable replication of the final analysis. CONCLUSION: When compared with trials using the rolling 6 design, those implementing the TITE-CRM design exhibited notable deficiencies in reporting essential details necessary for reproducibility. Inadequate reporting quality of advanced model-based trial designs hinders their credibility. We provide recommendations that can improve transparency, reproducibility, and accurate interpretation of the results for such designs.

Assessing the reporting quality of early phase dose-finding trial protocols: a methodological review.

Background: The paradigm of early phase dose-finding trials has evolved in recent years. Innovative dose-finding designs and protocols which combine phases I and II are becoming more popular in health research. However, the quality of these trial protocols is unknown due to a lack of specific reporting guidelines. Here, we evaluated the reporting quality of dose-finding trial protocols. Methods: We conducted a cross-sectional study of oncology and non-oncology early phase dose-finding trial protocols posted on ClinicalTrials.gov in 2017-2023. A checklist of items comprising: 1) the original 33-items from the SPIRIT 2013 Statement and 2) additional items unique to dose-finding trials were used to assess reporting quality. The primary endpoint was the overall proportion of adequately reported items. This study was registered with PROSPERO (no: CRD42022314572). Finding: A total of 106 trial protocols were included in the study with the rule-based 3 + 3 being the most used trial design (39.6%). Eleven model-based and model-assisted designs were identified in oncology trials only (11/58, 19.0%). The overall proportion of adequately reported items was 65.1% (95%CI: 63.9-66.3%). However, the reporting quality of each individual item varied substantially (range 9.4%-100%). Oncology study protocols showed lower reporting quality than non-oncology. In the multivariable analysis, trials with larger sample sizes and industry funding were associated with higher proportions of adequately reported items (all p-values <0.05). Interpretation: The overall reporting quality of early phase dose-finding trial protocols is suboptimal (65.1%). There is a need for improved completeness and transparency in early phase dose-finding trial protocols to facilitate rigorous trial conduct, reproducibility and external review. Funding: None.

Early phase clinical trials in oncology: Realising the potential of seamless designs.

BACKGROUND: The pharmaceutical industry's productivity has been declining over the last two decades and high attrition rates and reduced regulatory approvals are being seen. The development of oncology drugs is particularly challenging with low rates of approval for novel treatments when compared with other therapeutic areas. Reliably establishing the potential of novel treatment and the corresponding optimal dosage is a key component to ensure efficient overall development. A growing interest lies in terminating developments of poor treatments quickly while enabling accelerated development for highly promising interventions. METHODS: One approach to reliably establish the optimal dosage and the potential of a novel treatment and thereby improve efficiency in the drug development pathway is the use of novel statistical designs that make efficient use of the data collected. RESULTS: In this paper, we discuss different (seamless) strategies for early oncology development and illustrate their strengths and weaknesses through real trial examples. We provide some directions for good practices in early oncology development, discuss frequently seen missed opportunities for improved efficiency and some future opportunities that have yet to fully develop their potential in early oncology treatment development. DISCUSSION: Modern methods for dose-finding have the potential to shorten and improve dose-finding and only small changes to current approaches are required to realise this potential.

A Bayesian adaptive design for dual-agent phase I-II oncology trials integrating efficacy data across stages.

Combination of several anticancer treatments has typically been presumed to have enhanced drug activity. Motivated by a real clinical trial, this paper considers phase I-II dose finding designs for dual-agent combinations, where one main objective is to characterize both the toxicity and efficacy profiles. We propose a two-stage Bayesian adaptive design that accommodates a change of patient population in-between. In stage I, we estimate a maximum tolerated dose combination using the escalation with overdose control (EWOC) principle. This is followed by a stage II, conducted in a new yet relevant patient population, to find the most efficacious dose combination. We implement a robust Bayesian hierarchical random-effects model to allow sharing of information on the efficacy across stages, assuming that the related parameters are either exchangeable or nonexchangeable. Under the assumption of exchangeability, a random-effects distribution is specified for the main effects parameters to capture uncertainty about the between-stage differences. The inclusion of nonexchangeability assumption further enables that the stage-specific efficacy parameters have their own priors. The proposed methodology is assessed with an extensive simulation study. Our results suggest a general improvement of the operating characteristics for the efficacy assessment, under a conservative assumption about the exchangeability of the parameters a priori.

Mechanism of lnRNA-ICL involved in lung cancer development in COPD patients through modulating microRNA-19-3p/NKRF/NF-κB axis.

The incidence of lung cancer (LC) in chronic obstructive pulmonary disease (COPD) patients is dozens of times higher than that in patients without COPD. Elevated activity of nuclear factor-k-gene binding (NF-κB) was found in lung tissue of patients with COPD, and the continuous activation of NF-κB is observed in both malignant transformation and tumor progression of LC, suggesting that NF-κB and its regulators may play a key role in the progression of LC in COPD patients. Here, we report for the first time that a key long non-coding RNA (lncRNA)-ICL involved in the regulation of NF-κB activity in LC tissues of COPD patients. The analyses showed that the expression of ICL significantly decreased in LC tissues of LC patients with COPD than that in LC tissues of LC patients without COPD. Functional experiments in vitro showed that exogenous ICL only significantly inhibited the proliferation, invasion and migration in primary tumor cells of LC patients with COPD compared to LC patients without COPD. Mechanism studies have shown that ICL could suppress the activation of NF-κB by blocking the hsa-miR19-3p/NKRF/NF-κB pathway as a microRNA sponge. Furthermore, In vivo experiments showed that exogenous ICL effectively inhibited the growth of patient-derived subcutaneous tumor xenografts (PDX) of LC patients with COPD and significantly prolonged the survival time of tumor-bearing mice. In a word, our study shows that the decrease of ICL is associated with an increased risk of LC in patients with COPD, ICL is not only expected to be a new therapeutic target for LC in COPD patients, but also has great potential to be used as a new marker for evaluating the occurrence, severity stratification and prognosis of LC in patients with COPD.

Mechanisms of Antitumor Invasion and Metastasis of the Marine Fungal Derivative Epi-Aszonalenin A in HT1080 Cells.

Epi-aszonalenin A (EAA) is an alkaloid that is isolated and purified from the secondary metabolites of coral symbiotic fungi and has been shown to have good atherosclerotic intervention activity and anti-angiogenic activity in our previous studies. In the present study, antiangiogenic activity was used as a basis of an intensive study of its mechanism of action against tumor metastasis and invasion. Invasive metastatic pairs are a hallmark of malignancy, and the dissemination of tumor cells is the most dangerous process in the development of tumors. The results of cell wound healing and the Transwell chamber assay showed that EAA interfered well with PMA-induced migration and invasion of HT1080 cells. Western blot and the ELISA assay showed that EAA decreased MMPs and vascular endothelial growth factor (VEGF) activity and inhibited the expression of N-cadherin and hypoxia-inducible factor-1α (HIF-1α) by regulating the phosphorylation of downstream mitogen-activated protein kinase (MAPK), PI3K/AKT, and NF-κB pathways. Simultaneous molecular docking results revealed that the mimic coupling between the EAA and MMP-2/-9 molecules formed a stable interaction. The results of this study provide a research basis for the inhibition of tumor metastasis by EAA, and together with previous studies, confirm the potential pharmacology and drug potential for this class of compound for application in angiogenesis-related diseases and further improve the availability of coral symbiotic fungi.

Amelioration of atherosclerosis in ox-LDL induced HUVEC by sulfated polysaccharides from Gelidium crinale with antihypertensive activity.

Red algal polysaccharide is a good potential medical resource. Different red algal polysaccharides have different structural characteristics and rich biological activities. Previous studies have identified some structural information of sulfated polysaccharide (GNP, 25.8 kDa) from red algae, Gelidium crinale and found that GNP has excellent anti-inflammatory, antioxidant and anti-tumor activities. On this basis, this study investigated the effect of GNP on atherosclerosis, which is closely related to antioxidant and anti-inflammatory mechanisms and usually coexists and interacts with hypertension. This study investigated the inhibitory activity of GNP on angiotensin-converting enzyme (ACE) and its mechanism on oxidized low-density lipoprotein (ox-LDL)-induced HUVEC atherosclerosis. The results showed that GNP inhibits the up-regulation of cell adhesion molecules and oxidized low-density lipoprotein receptor-1 (LOX-1). GNP can regulate mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-κB) and PI3K/AKT signal pathways, inhibit apoptosis, invasion and migration. Meanwhile, GNP (IC50 = 269.2 µg/mL) antagonizes ACE by competitive binding mode, and it can reduce systolic blood pressure (SBP) of spontaneously hypertensive rats (SHR). It provides a theoretical basis for GNP as a potential substance for the prevention and treatment of atherosclerosis.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. SIGNIFICANCE: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

The pro-invasive factor COL6A2 serves as a novel prognostic marker of glioma.

Background: Glioma is an incurable malignant lesion with poor outcome characterized by easy recurrence after surgery with or without radiotherapy and chemotherapy. Studies have shown that COL6A2 is closely related to the tumorigenesis and development of a variety of tumors. However, the role of COL6A2 in glioma and the relationship between COL6A2 and tumor infiltrating immune cells remain unclear. Methods: Western blot, real-time PCR, a tissue microarray and immunohistochemistry were applied to detect COL6A2 mRNA and protein amounts in glioma, and all experiments were repeated three times. A tissue microarray of glioma samples was used for prognostic analysis. Detection of COL6A2 co-expression with immune genes using immunohistochemical methods, and tumor modeling using nude mice for prevention and treatment studies. Based on the mRNA expression of COL6A2, patients with glioma in TCGA were divided into the low and high COL6A2 expression groups, and GO and KEGG pathway analyses were performed. A PPI network was constructed using STRING, and the associations of COL6A2 with tumor-infiltrating immune cells and immune genes were analyzed in the CIBERSORT and TISIDB databases. COL6A2 mRNA and protein amounts were increased in glioma. Results: Multiple-database and tissue microarray analyses showed that COL6A2 expression in glioma was associated with poor prognosis, Tissue microarray showed that COL6A2 was the highest expressed in WHO IV and significantly higher in TCGA-GBM than in TCGA-LGG. Immunohistochemistry can well demonstrate the co-expression of COL6A2 with immune genes in a tumor model established in nude mice, showing that interference with COL6A2 expression may have an inhibitory effect on tumors. The mRNA expression of COL6A2 was involved in 22 KEGG pathways, and GSEA analysis showed that 28 and 57 gene sets were significantly enriched at nominal p values <0.01 and <0.05, respectively, protein network revealed a tight interaction between COL6A2 and SPARC. The CIBERSORT database indicated that COL6A2 was correlated with 15 types of tumor-infiltrating immune cells, including M2 macrophages, CD8 T cells, neutrophils, gamma delta T cells, activated CD4 memory T cells, follicular helper T cells, M0 macrophages, M1 macrophages, regulatory T cells (Tregs), activated NK cells, eosinophils, activated mast cells, monocytes, activated dendritic cells, and resting CD4 memory T cells. The TISIDB database indicated that COL6A2 was significantly correlated with lymphocytes such as regulatory T cell, Type 17 T helper cell, Type 1 T helper cell, and immunomodulatory genes. In addition, COL6A2-related immune regulatory genes show that most immune regulatorygenes have prognostic value for glioma, and high-risk immune genes are notconducive to the survival of glioma patients. Conclusions: COL6A2-related immune regulatory genes show that most immune regulatory genes have prognostic value for glioma, and high-risk immune genes are not conducive to the survival of glioma patients. COL6A2 may be a novel potential prognostic biomarker of glioma and associated with tumor-infiltrating immune cells in the tumor microenvironment, and interference with COL6A2 expression can inhibit tumor growth, which suggests COL6A2 as a potential target for future treatment.

miR-100-5p Is a Novel Biomarker That Suppresses the Proliferation, Migration, and Invasion in Skin Cutaneous Melanoma.

Objective: To better understand the role and underlying mechanisms of SKCM, we conducted bioinformatics analysis and in vivo experiments. Results: We found its role as a tumor suppressor gene in SKCM and its effect on prognosis. In addition, this study found that miR-100-5p had a bidirectional effect on SKCM microenvironment. After exploring the relationship between the two, it was found that tumors with intermediate miR-100-5p expression had the highest level of immune cell infiltration. In addition, the value of miR-100-5p was assessed by survival analysis, univariate Cox regression analysis, and nomogram prognostic prediction. Finally, we constructed a regulatory network to illustrate the regulatory relationship of miR-100-5p. Conclusions: In conclusion, the antitumor effect of miR-100-5p is revealed, and the present study is followed by a discussion of its molecular regulatory network, followed by novel insights into SKCM therapy.

A novel melanoma prognostic model based on the ferroptosis-related long non-coding RNA.

Ferroptosis is an iron-dependent programmed cell death related to the biological process of many kinds of tumors. Long noncoding RNAs (LncRNA) have been found to play essential roles in the tumor, and their functions in the ferroptosis of tumor cells have been partially discovered. However, there is no summary of ferroptosis-related LncRNA and its functions in melanoma. In the present study, we aim to explore the expression profile of ferroptosis-related LncRNA genes and their value in melanoma prognosis by bioinformatics analysis. The expression of ferroptosis-related gene (FRG) from melanoma clinical data was extracted based on the Cancer Genome Atlas (TCGA) database. By screening the RNA expression data of 472 cases of melanoma and 810 cases of normal skin, eighteen ferroptosis-related differential genes were found to be related to the overall survival rate. Furthermore, 384 ferroptosis-related LncRNAs were discovered through constructing the mRNA-LncRNA co-expression network, and ten of them were found with prognostic significance in melanoma by multivariate Cox analysis. Risk assessment showed that the high expression of LncRNA00520 is associated with poor prognosis, while the increased expression of the other LncRNA is beneficial to the prognosis of patients with melanoma. From univariate and multivariate Cox regression analysis, there were ten ferroptosis-related LncRNA risk models towards to be significant independent prognostic factors for patients with melanoma and valuable predictive factors for overall survival (OS)(P<0.05). The ROC curve further suggested that the risk score has relatively reliable predictive ability (AUC=0.718). The protein level of ferroptosis-related genes was verified by the HPA database and IHC test, leading to the discovery that the expressions of ALOX5, PEBP1, ACSL4, and ZEB1 proteins up-regulated in tumor tissues, and existed differences between tumor tissues and normal tissues. In conclusion, we identified ten ferroptosis-related LncRNA and constructed a prognosis model base.

Design and analysis of umbrella trials: Where do we stand?

Background: The efficiencies that master protocol designs can bring to modern drug development have seen their increased utilization in oncology. Growing interest has also resulted in their consideration in non-oncology settings. Umbrella trials are one class of master protocol design that evaluates multiple targeted therapies in a single disease setting. Despite the existence of several reviews of master protocols, the statistical considerations of umbrella trials have received more limited attention. Methods: We conduct a systematic review of the literature on umbrella trials, examining both the statistical methods that are available for their design and analysis, and also their use in practice. We pay particular attention to considerations for umbrella designs applied outside of oncology. Findings: We identified 38 umbrella trials. To date, most umbrella trials have been conducted in early phase settings (73.7%, 28/38) and in oncology (92.1%, 35/38). The quality of statistical information available about conducted umbrella trials to date is poor; for example, it was impossible to ascertain how sample size was determined in the majority of trials (55.3%, 21/38). The literature on statistical methods for umbrella trials is currently sparse. Conclusions: Umbrella trials have potentially great utility to expedite drug development, including outside of oncology. However, to enable lessons to be effectively learned from early use of such designs, there is a need for higher-quality reporting of umbrella trials. Furthermore, if the potential of umbrella trials is to be realized, further methodological research is required.

Cell origin and genome profile difference of penoscrotum invasive extramammary Paget disease compared with its in situ counterpart.

Penoscrotum extramammary Paget disease (pEMPD) is a rare cutaneous carcinoma with an unknown cell origin. pEMPD always presents as a tumor in situ with an indolent process, whereas some progress into invasive forms with more aggressive behavior. The in situ and invasive cases display different morphologies and biological behavior, and thus far, a relationship between these two components has not been demonstrated. Immunohistochemistry was used to disclose the immunotype of pEMPD, and the results revealed that invasive/in situ pEMPD possessed with some identical immunophenotypes such as CK7, P63, and CK10, which inferred the clonal relatedness. The variable expressions of GCDFP-15 and carcino embryonic antigen hinted that tumor cell origin might be an epidermal sweat gland in epiderma. In our cohort, invasive pEMPD presented increased expression of androgen receptor and decreased MUC5CA expression, and these two changes might bring to the shift of invasive phenotype. To better understanding the relationship between these distinct tumor forms, we performed whole exome sequencing testing to evaluate overlapping genomic alterations of six paired invasive/in situ pEMPDs. The results showed that missense mutation was the predominant mutation type, and C>T transition accounted for 65.1% in all SNP mutation. Among the top 20 differential genes obtained from the six paired invasive/in situ pEMPD analysis, MUC4 (one missense, one in frame del, and one multi-hit), AHNAK2 (two missense and one multi-hit), DOT1L (two missense and one multi-hit), and FRG1 (two missense and one-multi hit) mutations were most enriched in invasive pEMPDs, which postulated that these genes may play roles in the disease progression.

Comprehensive analysis of transcriptome characteristics and identification of TLK2 as a potential biomarker in dermatofibrosarcoma protuberans.

Background: Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous sarcoma characterized by local invasion and recurrence. RNA sequencing (RNA-seq) allows the qualification of cellular RNA populations and provides information on the transcriptional state. However, few studies have comprehensively analyzed DFSP transcriptional data. Methods: Fourteen DFSP samples with paired non-neoplastic soft tissue from Chinese patients undergoing Mohs micrographic surgery were used for RNA-seq analysis. Differential expression analysis and enrichment analysis for RNA-seq data were performed to identify fusion genes, biomarkers, and microenvironment characteristics of DFSP. Results: This study systemically describes the transcriptomic characteristics of DFSP. First, we performed gene fusion analysis and identified a novel FBN1-CSAD fusion event in a DFSP patient with fibrosarcomatous transformation. Then, we identified TLK2 as a biomarker for DFSP based on functional enrichment analysis, and validated its accuracy for diagnosing DFSP by immunohistochemical staining and joint analysis with public data. Finally, microenvironment analysis described the infiltration characteristics of immune and stromal cells in DFSP. Conclusion: This study demonstrates that RNA-seq can serve as a promising strategy for exploring molecular mechanisms in DFSP. Our results provide new insights into accurate diagnosis and therapeutic targets of DFSP.

A-Kinase Interacting Protein 1 Knockdown Restores Chemosensitivity via Inactivating PI3K/AKT and ß-Catenin Pathways in Anaplastic Thyroid Carcinoma.

Background: A-kinase interacting protein 1 (AKIP1) promotes tumor progression and chemoresistance in several malignancies; meanwhile, it is related to higher tumor size and recurrence risk of papillary thyroid carcinoma, while the role of AKIP1 in anaplastic thyroid carcinoma (ATC) is unclear. The aim of this study is to explore the effect of AKIP1 knockdown on cell malignant behaviors and doxorubicin resistance in ATC. Methods: AKIP1 knockdown was conducted in ATC cell lines (8505C and CAL-62 cells) by siRNA; then, cell viability, apoptosis, invasion, PI3K/AKT and ß-catenin pathways, and doxorubicin sensitivity were detected. Subsequently, doxorubicin-resistant 8505C cells (8505C/Dox) were established. Additionally, AKIP1 was modified in 8505C and 8505C/Dox cells that underwent doxorubicin treatment by siRNA or overexpression plasmid, followed by cellular function and pathway detection. Results: AKIP1 was elevated in FRO, 8505C, CAL-62, and KHM-5M cells compared to control cells (all p < 0.05). Subsequently, AKIP1 knockdown elevated apoptosis, inhibited viability and invasion, and inactivated PI3K/AKT and ß-catenin pathways in 8505C and CAL-62 cells (all p < 0.05). AKIP1 knockdown decreased relative cell viability in doxorubicin-treated 8505C and CAL-62 cells; then, AKIP1 was elevated in 8505C/Dox cells compared to 8505C cells (all p < 0.05). Furthermore, AKIP1 knockdown restored doxorubicin sensitivity (reflected by decreased cell viability and invasion, and increased apoptosis), but inactivated PI3K/AKT and ß-catenin pathways in doxorubicin-treated 8505C/Dox cells. However, AKIP1 overexpression presented an opposite effect on these functions and pathways in doxorubicin-treated 8505C cells. Conclusion: AKIP1 knockdown decreases cell survival and invasion while promoting sensitivity to doxorubicin via inactivating PI3K/AKT and ß-catenin pathways in ATC.

A Sulfated Polysaccharide from Red Algae (Gelidium crinale) to Suppress Cells Metastasis and MMP-9 Expression of HT1080 Cells.

Sulfated polysaccharides from red algae have a variety of biological activities, especially antitumor activities. Matrix metalloproteinase-9 (MMP-9) is a proteolytic metalloenzyme that degrades the central part of the extracellular matrix (ECM) and promotes tumor metastasis. In this research, we have investigated the influence and mechanism of GNP (sulfated polysaccharide from Gelidium crinale) on tumor metastasis and MMP-9 expression of human fibrosarcoma (HT1080) cells. The results inflected that the concentration of GNP below 100 µg/mL has no toxicity to HT1080 cells, but showed excellent activity in inhibiting cells migration and invasion. In addition, GNP effectively inhibits the mRNA of MMP-9 and reduces its expression and activity by regulating nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPK) and mTOR/PI3K/Akt signaling pathways. GNP has great potential as MMP-9 inhibitor and could be developed as a functional food or drug to prevent tumor metastasis.