Proliferation-related Pathways Research Articles

Anti-Oxidant and Anti-Inflammatory Effects of Astaxanthin on Gastrointestinal Diseases.

A moderate amount of reactive oxygen species (ROS) is produced under normal conditions, where they play an important role in cell signaling and are involved in many aspects of the immune response to pathogens. On the other hand, the excessive production of ROS destructs macromolecules, cell membranes, and DNA, and activates pro-inflammatory signaling pathways, which may lead to various pathologic conditions. Gastrointestinal (GI) mucosa is constantly exposed to ROS due to the presence of bacteria and other infectious pathogens in food, as well as alcohol consumption, smoking, and the use of non-steroidal anti-inflammatory drugs (NSAID). Prolonged excessive oxidative stress and inflammation are two major risk factors for GI disorders such as ulcers and cancers. Bioactive food compounds with potent anti-oxidant and anti-inflammatory activity have been tested in experimental GI disease models to evaluate their therapeutic potential. Astaxanthin (AST) is a fat-soluble xanthophyll carotenoid that is naturally present in algae, yeast, salmon, shrimp, and krill. It has been shown that AST exhibits protective effects against GI diseases via multiple mechanisms. Residing at the surface and inside of cell membranes, AST directly neutralizes ROS and lipid peroxyl radicals, enhances the activity of anti-oxidant enzymes, and suppresses pro-inflammatory transcription factors and cytokines. In addition, AST has been shown to inhibit cancer cell growth and metastasis via modulating cell proliferation-related pathways, apoptosis, and autophagy. Considering the potential benefits of AST in GI diseases, this review paper aims to summarize recent advances in AST research, focusing on its anti-oxidant and anti-inflammatory effects against gastric and intestinal ulcers and cancers.

Identification of hub genes in AR-induced tamoxifen resistance in breast cancer based on weighted gene co-expression network analysis.

Approximately 30% of patients with oestrogen receptor (ER)-positive breast cancer (BC) exhibit intrinsic or recurrent resistance to tamoxifen (TAM) adjuvant endocrine therapy. The androgen receptor (AR) is expressed in about 90% of ER-positive patients. Our previous studies found that BC patients with an AR:ER expression ratio ≥ 2.0 are more susceptible to TAM resistance. However, the specific mechanism by which a high AR:ER ratio promotes TAM resistance remains unknown. RNA sequencing was performed on 10 cases of BC tissues with AR:ER ratios ≥ 2.0 and 3 cases with AR:ER ratios < 2.0. We then compared our data with the screened TAM-resistant and TAM-sensitive cases from the TCGA BC database. Bioinformatics methods were used to screen differentially expressed genes (DEGs) and to perform gene enrichment analysis. Weighted correlation network analysis (WGCNA) was used to screen hub genes in the AR-induced TAM resistance process. PAM50 analysis showed that the molecular phenotype of BC patients with AR:ER ratios ≥ 2.0 was similar to that of triple-negative breast cancer (TNBC), whereas the BC samples with AR:ER ratios < 2.0 were classified as the luminal subtype. Among the AR:ER ratio ≥ 2.0 and AR:ER < 2.0 BC tumours, 1855 DEGs were identified. Gene enrichment analysis showed that DEGs were enriched mainly in proliferation-related molecular pathways, such as the cell cycle, necroptosis, metabolic pathways and DNA replication. WGCNA analysis showed that SEC14L2, RIIAD1, STC2 and MAGEA6 served as hub genes in AR-induced TAM resistance and were associated with BC survival prognosis in the TCGA cohort. A high AR:ER expression ratio is a biomarker for patients who might develop TAM resistance, and AR expression seems to be a possible mechanism of resistance to endocrine therapy.

Identification of key genes associated with cancer stem cell characteristics in Wilms’ tumor based on bioinformatics analysis

Nephroblastoma, also known as Wilms' tumor (WT), remains one of the major causes of tumor-related deaths worldwide in children. Cancer stem cells (CSCs) are considered to be the main culprits in cancer resistance and disease recurrence, which are reported in multiple types of tumors. However, the research on CSCs in WT is limited. Therefore, our study aimed to identify the key genes related to CSCs in WT to provide new ideas for treating WT. The RNA-seq and clinical data of WT samples were obtained from the University of California Santa Cruz (UCSC) Xena database, which included 120 WT and six para-cancerous tissues. The mRNA stemness index (mRNAsi) based on mRNA expression was calculated to evaluate tumor stem cell characteristics in WT patients. A Kaplan-Meier (KM) analysis was performed to explore the clinical characteristics of the mRNAsi in WT. A weighted gene co-expression network analysis (WGCNA) was used to identify the key modules and genes related to the mRNAsi. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to explore the signaling pathways based on the key genes. The expression levels of the key genes were validated by the Gene Expression Omnibus (GEO) database. Further, the important upstream genes were identified by DisNor and gene co-expression analyses. The mRNAsi was significantly upregulated in WT (P=7.2e-05) and showed an upward trend in line with the pathological stage. Patients with lower mRNAsi scores had better overall survival (OS) than those with higher mRNAsi scores (P=0.0087). Eleven genes were defined as the key genes associated with the mRNAsi based on our WGCNA analysis [cor.MM (correlation. Module membership) >0.8 and cor.GS (correlation. Gene significance) >0.45] and were closely related to cell proliferation-related signaling pathways (P<0.05). Moreover, using protein interaction analysis, we identified ATM and CDKN1A as the key upstream regulatory genes of the 11 key genes. Our study showed that the mRNAsi score was a potential prognostic factors in WT and identified the upstream genes ATM and CDKN1A and 11 genes closely related to the mRNAsi, which may provide new insights for CSC-targeted therapy in WT and improve clinical outcomes for WT patients.

Thyroid Hormone Receptor β Knockdown Reduces Suppression of Progestins by Activating the mTOR Pathway in Endometrial Cancer Cells.

Progestin resistance is a major obstacle to conservative therapy in patients with endometrial cancer (EC) and endometrial atypical hyperplasia (EAH). However, the related inducing factor is yet unclear. In this study, thyroid hormone and its receptor α (TRα) and β (TRβ) of patients were assayed. THRB-silenced RL95-2 and KLE EC cells were cultured to investigate the response of progestins. Transcriptomics and Western blotting were performed to investigate the changes in signaling pathways. We found that THRB, rather than THRA, knockdown promoted the viability and motilities of RL95-2 cells but not KLE cells. The suppressive effect of progestins on cell growth and motility significantly decreased in THRB-silenced RL95-2 cells. Multiple proliferation-related signaling pathways were enriched, and the activities of mammalian targets of rapamycin (mTOR)/4e-binding protein 1 (4EBP1)/eukaryotic translation initiation factor 4G (eIF4G) rather than phosphorylated protein kinase B (Akt) were remarkably boosted. Progestin treatment enhanced the effects, and the augmentation was partially abated on supplementation with T3. In THRB-knockdown KLE cells, the progestins-activated partial signaling pathway expression (either mTOR or eIF4G), and supplementation with T3 did not induce noticeable alterations. The serum levels of triiodothyronine (T3) were significantly lower in patients with EC compared with healthy women. A strong expression of TRβ was observed in most patients with EC and EAH sensitive to progestin treatment. In contrast, TRα positive expression was detected in less than half of the patients sensitive to progestin therapy. In conclusion, THRB knockdown enhanced the viability and motility of type I EC cells and attenuated the suppressive effects of progestins by activating the mTOR-4EBP1/eIF4G pathway. Lower expression of THRB is likely correlated with progesterone resistance.

Machine learning-based prognosis signature for survival prediction of patients with clear cell renal cell carcinoma

The sole clinicopathological characteristic is not enough for the prediction of survival of patients with clear cell renal cell carcinoma (ccRCC). However, the survival prediction model constructed by machine learning technology for patients with ccRCC using clinicopathological features is rarely reported yet. In this study, a total of 5878 patients diagnosed as ccRCC from four independent patient cohorts were recruited. The least absolute shrinkage and selection operator analysis was implemented to identify optimal clinicopathological characteristics and calculate each coefficient to construct the prognosis model. In addition, weighted gene co-expression network and gene enrichment analysis associated with risk score were also carried out. Three clinicopathologic features were selected for the construction of the prognosis risk score model as the prognostic factors of ccRCC, including tumor size, tumor grade, and tumor stage. In the CPTAC (Clinical Proteomic Tumor Analysis Consortium) cohort, the General cohort, the SEER (Surveillance, Epidemiology, and End Results) cohort, and the Huashan cohort, patients with high-risk score had worse clinical outcomes than patients with low-risk score (hazard ratio 5.15, 4.64, 3.96, and 5.15, respectively). Further functional enrichment analysis demonstrated that our machine learning-based risk score was significantly connected with some cell proliferation-related pathways, consisting of DNA repair, cell division, and cell cycle. In summary, we developed and validated a machine learning-based prognosis prediction model, which might contribute to clinical decision-making for patients with ccRCC.

RNA 5-methylcytosine status is associated with DNMT2/TRDMT1 nuclear localization in osteosarcoma cell lines

Osteosarcoma (OS) is a pediatric malignant bone tumor with unsatisfying improvements in survival rates due to limited understanding of OS biology and potentially druggable targets. The present study aims to better characterize osteosarcoma U-2 OS, SaOS-2, and MG-63 cell lines that are commonly used as in vitro models of OS. We focused on evaluating the differences in cell death pathways, redox equilibrium, the activity of proliferation-related signaling pathways, DNA damage response, telomere maintenance, DNMT2/TRDMT1-based responses and RNA 5-methylcytosine status. SaOS-2 cells were characterized by higher levels of superoxide and nitric oxide that promoted AKT and ERK1/2 activation thus modulating cell death pathways. OS cell lines also differed in the levels and localization of DNA repair regulator DNMT2/TRDMT1. SaOS-2 cells possessed the lowest levels of total, cytoplasmic and nuclear DNMT2/TRDMT1, whereas in MG-63 cells, the highest levels of nuclear DNMT2/TRDMT1 were associated with the most pronounced status of RNA 5-methylcytosine. In silico analysis revealed potential phosphorylation sites at DNMT2/TRDMT1 that may be related to the regulation of DNMT2/TRDMT1 localization. We postulate that redox homeostasis, proliferation-related pathways and DNMT2/TRDMT1-based effects can be modulated as a part of anti-osteosarcoma strategy reflecting diverse phenotypic features of OS cells.

Amphiregulin Supplementation During Pig Oocyte In Vitro Maturation Enhances Subsequent Development of Cloned Embryos by Promoting Cumulus Cell Proliferation.

The oocyte in vitro maturation (IVM) technique is important in animal husbandry, biomedicine, and human-assisted reproduction. However, the developmental potential of in vitro matured oocytes is usually lower than that of in vivo matured (IVVM) oocytes. Amphiregulin (AREG) is an EGF-like growth factor that plays critical roles in the maturation and development of mammalian oocytes. This study investigated the effects of AREG supplementation during pig oocyte IVM on the subsequent development of cloned embryos. The addition of AREG to pig oocyte IVM medium improved the developmental competence of treated oocyte-derived cloned embryos by enhancing the expansion and proliferation of cumulus cells (CCs) during IVM. The positive effect of AREG on enhancing the quality of IVVM pig oocytes might be due to the activation of proliferation-related pathways in CCs by acting on the AREG receptor. The present study provides an AREG treatment-based method to improve the developmental competence of cloned pig embryos.

Abstract 6091: Evolutionary characterisation of lung adenocarcinoma pathological subtypes in TRACERx

Abstract Background: Lung adenocarcinoma (LUAD) is a morphologically and genetically diverse disease. The prognostic impact of LUAD histological patterns have been described, such as solid growth pattern and poor outcomes, though their underlying biology is poorly understood. Furthermore, the genomic characteristics and evolutionary constraints in relation to the inter- and intra- tumoral variance of histological patterns in primary and metastatic disease are unknown. Methods: Pathological classification of 246 patients with LUAD from the TRACERx 421 cohort was performed at the whole tumor (diagnostic samples) and multi-regional sample level (matched for tumor whole exome sequencing and RNA sequencing). Circulating tumor DNA (ctDNA) data was also integrated to determine the relationship between pathological subtypes and ctDNA detection. Results: Chromosomal instability, characterized by fraction of the genome affected by subclonal copy number alterations was significantly correlated with proportion of high-grade patterns, namely solid, cribriform and micropapillary (Spearman’s Rho 0.27, p&amp;lt;0.001). Analysis of somatic copy number alterations (SCNAs) and driver mutation profiles showed that specific SCNAs were associated with a predominant growth pattern, such as 3q arm gains in predominantly cribriform and solid pattern tumors. Multiregional analysis of tumors with mixed patterns showed higher grade regions to be associated with a higher frequency of LOH and expression of proliferation-related pathway genes, suggesting intra-tumoral sequential evolution from low to high grade growth patterns. No recurrent subclonal mutations or SCNAs were found to associate with progression from low to high grade patterns. The growth pattern in metastatic tumors tended to show similar or a higher-grade pattern compared with primary tumor regions harboring metastasizing clones (seeding regions). The growth pattern of the seeding regions in the primary tumor was not necessarily higher grade compared with their non-seeding counterparts. Finally, the proportion of solid pattern in the primary tumor and the presence of necrosis were found to be strongly associated with pre-operative ctDNA detection, while histological ‘spread through air spaces’ (STAS) was identified in 92% (12/13) of pre-operative ctDNA-negative tumors that subsequently were associated with recurrence. Patients with both pre-operative detectable ctDNA and STAS had a particularly poor prognosis. Conclusion: These data reveal insights into the association between morphological and molecular heterogeneity in LUAD, describe key features of tumor evolutionary tendencies and demonstrate the utility of detailed tumor morphological assessment integrated with molecular characterization and ctDNA detection. Citation Format: Takahiro Karasaki, David A. Moore, Selvaraju Veeriah, Cristina Naceur-Lombardelli, Antonia Toncheva, Maise Al Bakir, Thomas B. Watkins, Oriol Pich, Alexander M. Frankell, Emilia Lim, Mark S. Hill, Kristiana Grigoriadis, Carlos Martinez-Ruiz, James R. Black, Clare Puttick, Dhruva Biswas, Ariana Huebner, Michelle Dietzen, Emma Colliver, Claudia Lee, Nnenna Kanu, Sadegh Mohammad Saghafinia, Francisco Gimeno Valiente, Christopher Abbosh, Crispin T. Hiley, Simone Zaccaria, Nicolai J. Birkbak, Allan Hackshaw, TRACERx Consortium, Teresa Marafioti, Roberto Salgado, John Le Quesne, Andrew G. Nicholson, Nicholas McGranahan, Charles Swanton, Mariam Jamal-Hanjani. Evolutionary characterisation of lung adenocarcinoma pathological subtypes in TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6091.

Signature based on B cells-related gene pairs could predict prognosis and immunotherapeutic response in NSCLC.

e21051 Background: Tumor-infiltrating B cells accompanying several widely used B cell-related biomarkers, such as CD19 and CD20, have inconsistent clinical prognostic values in non-small-cell lung cancer (NSCLC) patients. Considering only one B cell-related biomarker could not distinguish the anti-tumor and pro-tumor B cell subsets in tumor, we aimed to construct a more accurate B-cells related gene pairs (BRGPs) prognostic model and evaluate its potential predictive ability to immunotherapy in NSCLC patients. Methods: Using public single-cell RNA sequencing data, the B cells-related genes (BRGs) in NSCLC samples were identified. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were utilized to construct the BRGPs model which was not affected by the technical bias of different platforms. With no dependence upon specific gene expression levels, a novel signature based on BRGPs was established in this study. In addition, the prognostic value and immunotherapeutic response for this signature with regard to the TME components and potential molecular mechanism were explored. Results: Based on the TCGA database, we built a novel prognostic signature of 23 BRGPs comprising 28 unique BRGs. This risk model showed significant power in distinguishing good or poor prognosis and could serve as an independent prognostic factor for NSCLC patients in the TCGA cohort. The prognostic accuracy of the model was further verified in the GSE31210 dataset from the GEO database. Likewise, the prognostic value of the risk score for the BRGPs model was demonstrated in the GEO cohorts by the univariate and multivariate cox regression analysis. In addition, the risk model was significantly associated with sex, TNM stage, immune score, tumor purity and various tumor-infiltrating immune cells. GSEA analysis indicated that low-risk group enriched with several immune-related pathways, such as activation of immune response, antigen receptor mediated signaling pathway, B cell activation and B cell mediated immunity, whereas several proliferation-related pathways, such as nuclear chromosome segregation, sister chromatid segregation and mitotic sister chromatid segregation were most enriched in high-risk group. Besides, the tumor mutational burden (TMB) score rather than CD274(PD-L1 mRNA) expression was positively correlated with the risk score (P＜0.001; P = 0.94, respectively). NSCLC patients with high-risk exhibited significantly higher TMB score compared with low-risk patients (P &lt; 0.001). Correspondingly, we demonstrated that immune checkpoint blockade therapy may be more efficacious in high-risk group NSCLC patients according to TIDE method (P＜0.01). Conclusions: This novel BRGPs model can assess the prognosis of patients with NSCLC, and may be helpful to guide immune checkpoint inhibitors treatment in our clinical practice.

Identification and validation for biological characteristics and prognosis of tumor microenvironment subtypes in patients with lung adenocarcinoma.

e21006 Background: Immunotherapy has brought a great revolution to treatment of patients with lung adenocarcinoma (LUAD). However, the infiltrating cells in the tumor microenvironment (TME) could influence immunotherapy outcomes. This study aimed to investigate the TME subtypes based on 50 immune, stromal, and stem cell types in TME. Methods: The xCell algorithm was used to infer the enrichment scores in TME. We performed the k-mean consensus clustering to identify TME subtypes for LUAD patients. The ESTIMATE, ABSOLUTE, LUMP, and CPE algorithms were used to compare tumor purity and immune infiltration of TME subtypes. The genomic mutations and copy number variants were also calculated by “maftools” R package and GISTIC 2.0. We established a neural network model, based on the TCGA dataset and verified it in GEO datasets by the Kaplan-Meier curve and Cox regression analyses, to prove the stability of TME subtypes. Using prophetic algorithm, ridge regression model was established to predict the sensitivity of drugs in all datasets. In addition, TIDE score and submap analyses were used to speculate the potential response of patients in immunotherapy. Results: In our study, 50 types of cells, including immune (including lymphocyte and myeloid cells), stem, and stromal cells in TME, were included for analysis. Based on cell scores, 504 LUAD patients were divided into four different subtypes (C1, C2, C3 and C4). In univariable Cox regression, the hazard ratio (HR) value of C2 vs. C1 was 2.066 (95%CI: 1.313 - 3.250), the HR value of C3 vs. C1 was 1.821 (95%CI: 1.067- 3.108), and that of C4 vs. C1 was 2.313 (95%CI: 1.461 - 3.662). Then, a neural network model was established (the area under the curves in the testing cohort: 0.911) to validate our conclusions in external sets and similar results were observed. Through pseudotime trajectory, we discovered there were potential connections among clusters from C1 to C4. The C2 and C3 were the intermediate forms of C1 and C4, and thus, the survival, immunological, and metabolic status of C2 and C3 may be intermediate between C1 and C4. By comparing the cell infiltration and immune gene expression of the four clusters, we found that C1 and C3 may have a similar immune phenotype which was opposed to that of C2 and C4. Meanwhile, the enrichment scores of cell proliferation-related pathways were progressively increased in the four clusters, which may be related to survival outcomes of them. We noticed that C1 harbored a unique EGFR mutation compared to other clusters, which may be sensitive to erlotinib. Patients of C4 had lower TIDE scores, which indicated that these patients may get higher benefits from immunotherapy. Conclusions: In conclusion, we identified four TME subtypes with distinct clinical outcomes and biological features, including tumor immunity, TME status, tumor mutations, and responses to chemotherapy and immunotherapy.

Type 2 Diabetic Mellitus Inhibits Skin Renewal through Inhibiting WNT-Dependent Lgr5+ Hair Follicle Stem Cell Activation in C57BL/6 Mice.

Background Hair follicles are important accessory organs of the skin, and it is important for skin renewal and performs variety of important functions. Diabetes can cause several dermatoses; however, its effect on hair follicles is unclear. The purpose of this study was to investigate the effect of type II diabetes (T2DM) on the hair follicles of mice. Methods Seven-week-old male C57BL/6 littermate mice were divided into two groups. The treatment group was injected with streptozotocin (STZ) to induce T2DM, and the control group was parallelly injected with the same dose of buffer. Seven days after injection, the back is depilated to observe the hair follicle regeneration. Hair follicle regeneration was observed by naked eyes and HE staining. The proliferation of the skin cells was observed by PCNA and K14 staining. The altered genes were screened by RNA sequencing and verified by qRT-PCR. In addition, Lgr5 + GFP/mTmG transgenic mice were used to observe the effect of T2DM on Lgr5 hair follicle stem cells (HFSC). And the expression of WNT4 and WNT8A were measured by Western Blot. Results T2DM inhibited hair follicle regeneration. Compared to control mice, T2DM mice had smaller hair follicles, reduced skin thickness, and less expression of PCNA and K14. RNA sequencing showed that the two groups had significant differences in cell cycle and proliferation-related pathways. Compared with the control mice, the mRNA expression of Lgr4, Lgr5, Wnt4, and Wnt8a was decreased in the T2DM group. Moreover, T2DM inhibited the activation of Lgr5 HFSC and the expression of WNT4 and WNT8A. Conclusions T2DM inhibited hair follicle regeneration and skin cells proliferation by inhibiting WNT-dependent Lgr5 HFSC activation. This may be an important reason for the reduction of skin renewal ability and the formation of chronic wounds caused by diabetes. It is important for the treatment of chronic diabetic wounds and the development of tissue engineering.

Characterization of the Different Subtypes of Immune Cell Infiltration to Aid Immunotherapy.

Background?PD-1 ablation or PD-L1 specific monoclonal antibody against PD-1 can recruit the accumulation of functional T cells, leading to tumor rejection in the microenvironment and significantly improving the prognosis of various cancers. Despite these unprecedented clinical successes, intervention remission rates remain low after treatment, rarely exceeding 40%. The observation of PD-1/L1 blocking in patients is undoubtedly multifactorial, but the infiltrating degree of CD8+T cell may be an important factor for immunotherapeutic resistance. Methods:We proposed two computational algorithms to reveal the immune cell infiltration (ICI) landscape of 1646 lung adenocarcinoma patients. Three immune cell infiltration patterns were defined and the relative ICI scoring depended on principal-component analysis. Results:A high ICI score was associated with the increased tumor mutation burden and cell proliferation-related signaling pathways. Different cellular signaling pathways were observed in low ICI score subtypes, indicating active cell proliferation, and may be associated with poor prognosis. Conclusion:Our research identified that the ICI scores worked as an effective immunotherapy index, which may provide promising therapeutic strategies on immune therapeutics for lung adenocarcinoma.

Abstract P2-08-06: Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study

Abstract Introduction: Early lymph node (LN) metastasis often precedes systemic metastasis and corresponds with a 35% decrease in 10-year survival compared to patients without LN metastasis. Understanding biologic pathways involved in early LN metastasis could identify promising drug targets for early-stage breast cancer (EBC) treatment. While tumor size modestly correlates with the probability of clinical or occult axillary metastases, small T1 tumors are often found to have LN metastases, while many large T3 tumors are often LN negative. We compared large tumors without evidence of LN metastasis (pT2-3N0) and small tumors with LN metastasis (pT1N1-3) by whole transcriptome analysis to elucidate molecular biological differences associated with the presence or absence of early LN metastasis at diagnosis. Methods: The FLEX study enrolls patients with EBC who undergo standard of care MammaPrint (MP) and BluePrint (BP) testing, and consent to clinically annotated full transcriptome data collection. MP classifies tumors as having High Risk (HR) or Low Risk (LR) of distant recurrence. MP combined with BP subtype tumors as either Luminal A-type (MP LR; 56.8%), Luminal B-type (MP HR; 36.4%), HER2-type (1.5%), or Basal-type (5.3%). In our primary comparison, 355 patients had small, LN-positive tumors (pT1N1-3), and 235 had large, LN-negative tumors (pT2-3N0). Control groups consisted of small, LN-negative (pT1N0; n=1384) and large, LN-positive tumors (pT2-3N1-3; n=375). R package ‘limma’ was used for quantile normalization and differential gene expression (DGE) analyses. Differentially expressed genes (DEGs) with a false discovery rate &amp;lt; 0.05 and fold change &amp;gt; 1.4 were considered significant for this exploratory analysis. Gene set enrichment analysis (GSEA) was performed using the Hallmark gene sets (MSigDB). Results: Principal component analysis of each sample’s transcriptional profile revealed more gene profile differences between MP risk and BP subtype than by pathological stage. Overall, DGE analysis comparing pT2-3N0 to pT1N1-3 identified 30 DEGs; of which MUCL1 had a &amp;gt; 2-fold decrease. Within MP LR and BP Luminal A-type tumors, no DEGs were identified comparing pT2-3N0 to pT1N1-3. HR tumors had 73 DEGs (33 upregulated and 40 downregulated); two of 40 downregulated genes, including MUCL1, had &amp;gt; 2-fold change. Within Luminal B tumors, 34 DEGs were identified when comparing pT2-3N0 with pT1N1-3, although none had &amp;gt; 2-fold change. Basal and HER2 tumors were not analyzed due to small numbers. GSEA showed upregulated proliferation and oxidative phosphorylation pathways, and downregulated epithelial to mesenchymal transition (EMT) and immune signatures in pT2-3N0 relative to pT1N1-3 tumors overall, within HR, and Luminal B tumors. Conclusion: This study provides a foundation for understanding the mechanisms that promote LN metastasis. Studies have previously correlated LN metastasis with EMT, immune function, and gene overexpression (BCL2L1, AURKA, CDKN2A, MCL1, MYC). While we did not find significant differences in these genes, our results indicated similar differentially regulated pathways. Overall, we found most biological differences within MP HR and Luminal B tumors. Proliferation-related pathways were upregulated and EMT and immune-related pathways were downregulated in pT2-3N0 tumors compared with pT1N1-3 tumors, suggesting these pathways distinguish small, node-positive tumors from large, node-negative tumors and are involved in early LN metastasis. Interestingly, MUCL1 which has been reported to promote migration and invasion in breast cancer, was consistently downregulated in pT2-3N0 relative to pT1N1-3 tumors. Future studies will investigate antineoplastic therapies for EBC that modulate these dysregulated pathways to reduce early LN metastasis and subsequent systemic metastasis. Citation Format: Nibash Budhathoki, Faisal Fa'ak, Nina D'Abreo, Shubhada Dhage, Phyu Phyu Soe, Dennis Holmes, Josien Haan, Shiyu Wang, Patricia Dauer, Andrea Menicucci, William Audeh, Douglas K Marks. Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-06.

Long non-coding RNA RP11-342L8.2, derived from RNA sequencing and validated via RT-qPCR, is upregulated and correlates with disease severity in psoriasis patients.

This study aimed to explore the dysregulated long non-coding RNA (lncRNA) expression profile in psoriatic tissue vs. normal skin tissue via RNA sequencing (RNA-seq), then further sort candidate lncRNAs to be validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), in order to investigate the comprehensive linkage of lncRNA with psoriasis. Twenty-five psoriasis patients were consecutively enrolled, with their psoriatic and surrounding normal skin tissues obtained. Ten pairs of psoriatic and normal tissues were proposed to RNA-seq. Then, top 6 differentially expressed lncRNAs (DElncRNA) were sorted as candidate lncRNAs for validation by RT-qPCR in 25 pairs of samples. Principal component analysis (PCA) exhibited that lncRNA profile clearly distinguished psoriatic tissue from normal tissue, so did heatmap. Volcano plot disclosed 412 upregulated and 625 downregulated DElncRNAs in psoriatic tissue vs. normal tissue. Gene Ontology (GO) and Kyoko Encyclopedia of Genes and Genomes (KEGG) enrichment analyses exhibited that these DElncRNAs were mainly enriched in immune, inflammation, or proliferation-related biological processes and pathways such as neutrophil degranulation, regulation of immune response, positive regulation of cell proliferation, and MAPK signaling pathway. By RT-qPCR validation, lncRNAs RP11-22A3.2, RP11-342L8.2, and CTD-2006H14.2 were increased (all P < 0.001), while lncRNAs AP000442.4, CCDC144NL-AS1, and MIR663AHG were decreased (all P < 0.01) in psoriatic tissue vs. normal tissue. Interestingly, psoriatic lncRNA RP11-342L8.2 was also observed to positively correlated with psoriasis area and severity index (PASI) (r = 0.405, P = 0.045). Our present study exhibits some evidence for the landscape of lncRNAs implicated in psoriasis.

TNFR2 pathways are fully active in cancer regulatory T cells.

Tumor necrosis factor receptor 2 (TNFR2), a membrane-bound tumor necrosis factor receptor expressed by regulatory T cells (Tregs), participates in Treg proliferation. Although a specific TNFR2 pathway has been reported, the signaling mechanism has not been completely elucidated. This study sought to clarify TNFR2 signaling in human Tregs using amplicon sequencing and single-cell RNA sequencing to assess Tregs treated with a TNFR2 agonist antibody. Pathway enrichment analysis based on differentially expressed genes highlighted tumor necrosis factor α signaling via nuclear factor kappa B, interleukin-2 signal transducer and activator of transcription 5 signaling, interferon-γ response, and cell proliferation-related pathways in Tregs after TNFR2 activation. TNFR2-high Treg-focused analysis found that these pathways were fully activated in cancer Tregs, showing high TNFR2 expression. Collectively, these findings suggest that TNFR2 orchestrates multiple pathways in cancer Tregs, which could help cancer cells escape immune surveillance, making TNFR2 signaling a potential anticancer therapy target.

Proteome-wide analysis reveals molecular pathways affected by AgNP in a ROS-dependent manner

The use of mass spectrometry-based proteomics has been increasingly applied in nanomaterials risk assessments as it provides a proteome-wide overview of the molecular disturbances induced by its exposure. Here, we used this technique to gain detailed molecular insights into the role of ROS as an effector of AgNP toxicity, by incubating Bend3 cells with AgNP in the absence or presence of an antioxidant N-acetyl L-cystein (NAC). ROS generation is a key player in AgNP-induced toxicity, as cellular homeostasis was kept in the presence of NAC. By integrating MS/MS data with bioinformatics tools, in the absence of NAC, we were able to pinpoint precisely which biological pathways were affected by AgNP. Cells respond to AgNP-induced ROS generation by increasing their antioxidant pool, via NRF2 pathway activation. Additionally, cell proliferation-related pathways were strongly inhibited in a ROS-dependent manner. These findings reveal important aspects of the AgNP mechanism of action at the protein level.

Retracted: Lovastatin Inhibits RhoA to Suppress Canonical Wnt/β-Catenin Signaling and Alternative Wnt-YAP/TAZ Signaling in Colon Cancer

Statins are first-line drugs used to control patient lipid levels, but there is recent evidence that statin treatment can lower colorectal cancer (CRC) incidence by 50% and prolong CRC patient survival through mechanisms that are poorly understood. In this study, we found that the treatment of APCmin mice by the mevalonate pathway inhibitor lovastatin significantly reduced the number of colonic masses and improved hypersplenism and peripheral anemia. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis of colonic mass tissues showed a potent inhibitory effect in both Wnt/β-catenin signaling and YAP/TAZ signaling in the lovastatin treatment group. The results of our transcriptomic analyses in RKO indicated that lovastatin regulated several proliferation-related signaling pathways. Moreover, lovastatin suppressed important genes and proteins related to the canonical Wnt/β-catenin and alternative Wnt-YAP/TAZ signaling pathways in RKO and SW480 cells, and these effects were rescued by mevalonic acid (MVA), as confirmed through a series of Western blotting, RT-PCR, and reporter assays. Given that statins suppress oncogenic processes primarily through the inhibition of Rho GTPase in the mevalonate pathway, we speculate that lovastatin can inhibit certain Rho GTPases to suppress both canonical Wnt/β-catenin signaling and alternative Wnt-YAP/TAZ signaling. In RKO cells, lovastatin showed similar inhibitory properties as the RhoA inhibitor CCG1423, being able to inhibit β-catenin, TAZ, and p-LATS1 protein activity. Our results revealed that lovastatin inhibited RhoA activity, thereby suppressing the downstream canonical Wnt/β-catenin and alternative Wnt-YAP/TAZ pathways in colon cancer cells. These inhibitory properties suggest the promise of statins as a treatment for CRC. Altogether, the present findings support the potential clinical use of statins in non-cardiovascular contexts and highlight novel targets for anticancer treatments.

Presenilin is involved in larval-pupal metamorphosis development of Bombyx mori.

Disruption of the presenilin (ps) genes are the major genetic cause of familial Alzheimer's disease. The silkworm, Bombyx mori (B. mori), is an important model insect. The ps homologue gene in B. mori was identified and characterized. However, the role of ps in B. mori was poorly understood. Here, we found that Bmps was ubiquitously expressed in all the tested tissues during metamorphosis. In the current study, loss-of-function analysis of Bmps was performed by the binary transgenic CRISPR/cas9 system. Compared with the wild type, the developmental time of ∆Bmps animals were significantly delayed. In addition, ∆Bmps showed abnormal appendage including antenna, leg, wing and eye during pupal and adult stages. RNA-seq analysis indicated that apoptosis and proliferation related pathways were affected in ∆Bmps. Moreover, the Hippo pathway was affected by Bmps depletion in brain and wing disc. Our results suggest that PS is essential for maintaining the dynamic balance of apoptosis and proliferation during metamorphosis.

Nitric Oxide Dependent Metabolism Regulates the Proliferation and Differentiation of Stress Erythroid Progenitors

Infection and tissue damage induce inflammation, which increases myelopoiesis at the expense of steady state erythropoiesis. Stress erythropoiesis is induced to compensate for the loss of erythroid output until the inflammation is resolved and bone marrow erythropoiesis can resume. Steady state erythropoiesis constantly produces erythrocytes, while stress erythropoiesis generates a bolus of new erythrocytes through the rapid expansion of immature progenitor cells which is followed by the synchronous differentiation of progenitors. We hypothesized that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism.Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation.Notably, proliferating progenitor cells displayed low levels of the metabolite itaconate and decreased expression of Immunoresponsive gene 1 (Irg1), the enzyme that catalyzes itaconate synthesis from cis-aconitate. Further analysis showed that the addition of 4-Octyl itaconate (OI), a cell-permeable itaconate derivative, inhibited iNOS-derived NO production by activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn impaired progenitor expansion. These results indicate that itaconate production is inhibited to enable the accumulation of NO and the NO dependent metabolism required for progenitor cell proliferation during the initial expansion stage of stress erythropoiesis.In contrast, the transition to differentiation is marked by elevated itaconate synthesis, Nrf2 activation, and attenuated iNOS expression. We hypothesized that the inhibition of NO production alters metabolism and in concert with new cell signaling removes the NO-dependent inhibition of erythroid program, which allows the differentiation of progenitor cells. We tested this mechanism by examining the effects of iNOS inhibitors and mutants in iNOS, Irg1 and Nrf2 on progenitor cells isolated from differentiation cultures. iNOS deficiency led to the activation of erythroid transcriptional program, and increased numbers of mature progenitors as well as stress BFU-Es. In contrast, Irg1 and Nrf2 mutants showed impaired transition to erythroid differentiation, while they had elevated iNOS expression and NO production. Further analysis showed that treatment with either OI or iNOS inhibitor inhibited NO production in Irg1 and Nrf2 deficient progenitors, and consequently rescued the defects in erythroid differentiation.These data support a model in which inflammation inhibits steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis. Our work reveals a dynamic and tight coordination between pro-inflammatory signals and progenitor cell metabolism in regulating the proliferation and differentiation of stress erythroid progenitors, and highlights the therapeutic potential of targeting metabolic and inflammatory signaling pathways in inflammatory anemia. DisclosuresPaulson: Forma Therapeutics: Consultancy.

Self-renewal or quiescence? Orchestrating the fate of mesenchymal stem cells by matrix viscoelasticity via PI3K/Akt-CDK1 pathway

To control the fate of mesenchymal stem cells (MSCs) in a 3D environment by adjusting the mechanical parameters of MSC-loading scaffolds, is one of the hot topics in the field of regenerative biomaterials. However, a thorough understanding of the relevant MSCs behaviors affected by viscoelasticity, a dynamic physical parameter of scaffolds, is still lacking. Herein, we established an alginate hydrogel system with constant stiffness and tunable stress relaxation rate, which is a key parameter for the viscoelastic property of material. MSCs were cultured inside three groups of alginate hydrogels with various stress relaxation rates, and then RNA-seq analysis of cells was performed. Results showed that the change of stress relaxation rates of hydrogels regulated the most of the different expression genes of MSCs, which were enriched in cell proliferation-related pathways. MSCs cultured in hydrogels with fast stress relaxation rate presented a high self-renewal proliferation profile via activating phosphatidylinositol 3- kinase (PI3K)/protein kinase B (Akt) pathway. In contrast, a slow stress relaxation rate of hydrogels induced MSCs to enter a reversible quiescence state due to the weakened PI3K/Akt activation. Combined with a further finite element analysis, we speculated that the quiescence of MSCs could be served as a positive strategy for MSCs to deal with the matrix with a low deformation to keep stemness. Based on the results, we identified that stress relaxation rate of hydrogel was a potential physical factor of hydrogel to regulate the self-renewal or quiescence of MSCs. Thus, our findings provide a significant guiding principle for the design of MSCs-encapsulated biomaterials.