Colorectal cancer (CRC) is one of the most frequent gastrointestinal cancers worldwide, with high morbidity and mortality rates. Despite numerous attempts to identify prognostic markers for the CRC patients, the significance of the association of cellular proliferation markers with survival is controversial. Here we used immunohistochemistry to detect four markers of cellular proliferation expressed in primary CRC tissue specimens (n = 269) to assess their potential to serve as prognostic factors. CRC cells variably expressed phospho-histone H3 (PHH3) (range, 0-76 per high-powered field (HPF); median, 7 per HPF), cyclin A (CCNA) (range, 11.3-73.7%; median, 32%), geminin (GMNN) (range, 7.8-82.0%; median, 37.1%), and marker of proliferation Ki-67 (MKI67) (range, 4.9-96.6%; median, 49.6%). Among them, patients with PHH3-high (≥7 per HPF) tumors uniquely experienced significantly longer 5-year survival than those with PHH3-low (≤6 per HPF) (81.8% vs. 65.5%; P = 0.0047). Multivariable Cox hazards regression analysis identified PHH3-high (hazard ratio, 0.54; 95% confidence interval, 0.31-0.92; P = 0.025) as potential favorable factors. PHH3 levels inversely associated with pT stage (P < 0.0001) and were significantly and inversely associated with tumor diameter (ρ = -0.314, P < 0.0001). These findings support the use of PHH3 immunohistochemistry for predicting the prognoses of patients with CRC.

Keywords: colorectal cancer; immunohistochemistry; phospho-histone H3; prognostication; proliferation markers.

Estrogen-receptor-positive, and human epidermal growth factor receptor 2-negative (ER+HER2-) breast cancer accounts for ~60-70% of all cases of invasive breast carcinoma. High-grade ER+HER2- tumors respond poorly to endocrine therapy. In this study, we systematically analyzed clinical and multi-omics data to find potential strategies for personalized therapy of patients with high-grade ER+HER2- disease. Six different cohorts were analyzed, for which multi-omics data were available. Grade III ER+HER2- cases harbored higher proportions of large tumor size (>5cm), lymph nodes metastasis, chemotherapy use and luminal B subtypes defined by PAM50, as compared with grade I/II tumors. DNA methylation (HM450) data and methylation specific PCR indicated that the cg18629132 locus in the MKI67 promoter was hypermethylated in grade I/II cases and normal tissue, but hypomethylated in grade III cases or triple negative breast cancer (TNBC), resulting in higher expression of MKI67. Mutations in ESR1 and TP53 were detected in post-endocrine-treatment metastatic samples at a higher rate than in treatment-naive tumors in grade III cases. We identified 42 and 20 focal copy number events in non-metastatic and metastatic high-grade ER+HER2- cases, respectively, with either MYC or MDM2 amplification representing an independent prognostic event in grade III cases. Transcriptional profiling within grade III tumors, highlighted ER signaling downregulation and upregulation of immune-related pathways in non-luminal-like tumors defined by PAM50. Recursive partitioning analysis (RPA) was employed to construct a decision tree of an endocrine-resistant subgroup (GATA3-negative and AGR-negative) of two genes that was validated by immunohistochemistry in a Chinese cohort. All together, these data suggest that grade III ER+HER2- tumors have distinct clinical and molecular characteristics compared with low-grade tumors, particularly in cases with non-luminal-like biology. Due to the dismal prognosis in this group, clinical trials are warranted to test the efficacy of potential novel therapies.

Keywords: ER+HER2- breast cancer; endocrine-resistant subgroup; histologic grade; hypomethylated loci; multi-omics.

Glioblastoma (GBM) is the most aggressive and common brain cancer in adults with the lowest life expectancy. The current neuro-oncology practice has incorporated genes involved in key molecular events that drive GBM tumorigenesis as biomarkers to guide diagnosis and design treatment. This study summarizes findings describing the significant heterogeneity of GBM at the transcriptional and genomic levels, emphasizing 18 driver genes with clinical relevance. A pattern was identified fitting the stem cell model for GBM ontogenesis, with an upregulation profile for MGMT and downregulation for ATRX, H3F3A, TP53 and EGFR in the mesenchymal subtype. We also detected overexpression of EGFR, NES, VIM and TP53 in the classical subtype and of MKi67 and OLIG2 genes in the proneural subtype. Furthermore, we found a combination of the four biomarkers EGFR, NES, OLIG2 and VIM with a remarkable differential expression pattern which confers them a strong potential to determine the GBM molecular subtype. A unique distribution of somatic mutations was found for the young and adult population, particularly for genes related to DNA repair and chromatin remodelling, highlighting ATRX, MGMT and IDH1. Our results also revealed that highly lesioned genes undergo differential regulation with particular biological pathways for young patients. This multi-omic analysis will help delineate future strategies related to the use of these molecular markers for clinical decision-making in the medical routine.

Keywords: biomarkers; cancer genomics; diagnosis; glioma; tumour heterogeneity.

Background: Circular RNAs (circRNAs), a new class of regulatory noncoding RNAs, are involved in gene regulation and may play a role in cancer development. The aim of this study was to identify circRNAs involved in lung adenocarcinoma (LUAD) using bioinformatics analysis.

Methods: CircRNA (GSE101684, GSE101586), miRNA (GSE135918), and mRNA (GSE130779) microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed circRNAs (DECs), miRNAs (DEMs), and mRNAs (DEGs) in LUAD. Circinteractome and StarBase were used to predict miRNAs and mRNAs, respectively. A circRNA-miRNA-mRNA-ceRNA network was constructed. Patient survival was analyzed using UALCAN, and a sub-network was established. Real-time quantitative PCR (qRT-PCR) was used to verify the expressed of DECs between LUAD tissues and paired adjacent normal tissues.

Results: Hsa_circ_0072088 was identified as a differentially expressed (upregulated) circRNA in the two datasets. Intersection analysis identified hsa-miR-532-3p and hsa-miR-942 as the two miRNAs with the highest potential for binding to hsa_circ_0072088. Differential expression analysis and target gene prediction were performed to build a ceRNA network of hsa_circ_0072088 using Circinteractome/StarBase 3.0. Intersection analysis showed that TMEM52, IL24, POF1B, KIF1A, NHS, LBH, HIST2H2BE, ABCC3, PYCR1, CD79A, IGF2BP3, ANKRD17, GTSE1, MKI67, CLSPN, PLAU, LUC7L, MAGIX, GPATCH4, and ABAT were potential downstream mRNAs. The association between the expression level of 20 DEGs and LUAD patient survival was analyzed using UALCAN and GEPIA, which showed that IGF2BP3, MKI67, CD79A, and ABAT were related to patient survival. Hsa_circ_0072088 was verified upregulated by qRT-PCR.

Conclusion: The circRNA hsa_circ_0072088, the DEMs (hsa-miR-532-3p and hsa-miR-942-5p), and the DEGs (IGF2BP3, MKI67, CD79A, and ABAT) may serve as prognostic markers in LUAD.

Keywords: GEO; bioinformatics; ceRNA; circRNA; lung adenocarcinoma.

Patients with triple negative breast cancer (TNBC) have a poor prognosis. A novel prognostic biomarker may guide management by appropriately selecting patients for particular treatments. Peripheral blood neutrophil-to-lymphocyte ratio (NLR) was reported to associate with cancer progression, thus we hypothesized that intratumor genetic NLR will reflect tumor immune microenvironment (TIME) and breast cancer biology. The intratumoral genetic NLR previously defined as the ratio of CD66b (CEACAM8) and CD8 (CD8A) gene expressions was utilized to analyze total of 2,994 patients from METABRIC, TCGA, GSE21094, GSE22358, GSE25088, GSE32646, and GSE2603 cohorts. Intratumoral genetic NLR did not correlate with cancer stage nor clinical parameters of cancer cell proliferation such as Nottingham histological grade or MKI67 expression levels in neither the METABRIC or TCGA cohorts. Intratumoral genetic NLR-high breast cancer was not associated with pathologic complete response (pCR) after neoadjuvant chemotherapy in 5 independent cohorts with different regimens. Despite these results, intratumoral genetic NLR-high TNBC demonstrated worse disease-free, disease-specific, and overall survival. Intratumoral genetic NLR-low TNBC enriched multiple immune-related gene sets, was associated with higher favorable immune-related scores and with a favorable TIME, whereas no gene sets enriched to NLR-high TNBC. In conclusion, intratumoral genetic NLR-low TNBC was associated with favorable TIME and with better survival.

Keywords: GSEA; NLR; Neutrophil-to-lymphocyte ratio; TNBC; breast cancer; neoadjuvant chemotherapy; triple negative breast cancer; tumor immune microenvironment; xCell.

Recent studies have revealed that extensive heterogeneity of biological systems arises through various routes ranging from intracellular chromosome segregation to spatiotemporally varying biochemical stimulations. However, the contribution of physical microenvironments to single-cell heterogeneity remains largely unexplored. Here, we show that a homogeneous population of non-small-cell lung carcinoma develops into heterogeneous subpopulations upon application of a homogeneous physical compression, as shown by single-cell transcriptome profiling. The generated subpopulations stochastically gain the signature genes associated with epithelial-mesenchymal transition (EMT; VIM, CDH1, EPCAM, ZEB1, and ZEB2) and cancer stem cells (MKI67, BIRC5, and KLF4), respectively. Trajectory analysis revealed two bifurcated paths as cells evolving upon the physical compression, along each path the corresponding signature genes (epithelial or mesenchymal) gradually increase. Furthermore, we show that compression increases gene expression noise, which interplays with regulatory network architecture and thus generates differential cell-fate outcomes. The experimental observations of both single-cell sequencing and single-molecule fluorescent in situ hybridization agrees well with our computational modeling of regulatory network in the EMT process. These results demonstrate a paradigm of how mechanical stimulations impact cell-fate determination by altering transcription dynamics; moreover, we show a distinct path that the ecology and evolution of cancer interplay with their physical microenvironments from the view of mechanobiology and systems biology, with insight into the origin of single-cell heterogeneity.

Keywords: cell fate decision; cell volume; heterogeneity; mechanobiology; single cell.

RNA plays a central role in macromolecule biogenesis for various pathways, such as gene expression, ribosome biogenesis, and chromatin remodeling. However, RNA must be converted from its nascent to functional forms for that role. Here, we describe a large RNA metabolic network (RNAmetasome network) for macromolecule biogenesis in human cells. In HEK293T, the network consists of proteins responsible for gene expression, splicing, ribosome biogenesis, chromatin remodeling, and cell cycle. Reciprocal immunoprecipitations show that MKI67, GNL2, MDN1, and ELMSAN1 are core proteins of the network, and knockdown of either MKI67 or GNL2 affects the state of the other protein, MDN1, and some other network members. Furthermore, GNL2 knockdown retards cell proliferation. Several proteins of the RNAmetasome network are diminished in Hela.cl1, and this diminishment is associated with low expression of MDN1 and elevated MKI67 degradation. These results together suggest that the RNAmetasome network is present in human cells and associated with proliferation, and that MKI67, GNL2, and MDN1 play an important role in organizing the RNAmetasome network.

Inhibin-α, a member of transforming growth factor-β, is elevated in multiple tumors, but its specific roles are poorly understood. Here, we examined the feature of inhibin-α-expressing cells in ovarian tumors. Immunohistochemically, inhibin-α-expressing tumor cells were detected only in ovarian clear cell carcinoma (OCCC) among various types of ovarian tumors. By comparing the expression of inhibin-α and Ki-67, inhibin-α-expressing tumor cells were revealed to be less proliferative. When spheroids and chemoresistant cells were derived from OCCC cell lines, the expression level of inhibin-α was elevated, and that of an immature marker, aldehyde dehydrogenase, was also elevated. In consistent with this, inhibin-α expression was correlated with other immature markers, such as OCT3/4 and SOX2, and inversely correlated with proliferative marker MKI67 in public database on OCCC. Knockdown of inhibin-α in OCCC cell decreased chemoresistance. Moreover, prognostic analysis with 69 surgically resected OCCC cases revealed that the increased inhibin-α expression was an independent unfavorable prognostic factor. These findings suggested that inhibin-α-expressing subpopulation of OCCC tumor cells appeared to be less proliferative, immature, and angiogenic and to be related to acceleration of malignant progression.

Chronological skin ageing is an inevitable physiological process that results in thin and sagging skin, fine wrinkles, and gradual dermal atrophy. The main therapeutic approaches to soft tissue augmentation involve using dermal fillers, where natural fillers, such as autologous fibroblasts, are involved in generating dermal matrix proteins. The aim of this study was to determine the global transcriptome profile of three passages of dermal autologous fibroblasts from a male volunteer, focusing on the processes of the cell cycle and cell proliferation status to estimate the optimal passage of the tested cells with respect to their reimplantation. We performed K-means clustering and validation of the expression of the selected mRNA by qRT-PCR. Ten genes were selected (ANLN, BUB1, CDC20, CCNA2, DLGAP5, MKI67, PLK1, PRC1, SPAG5, and TPX2) from the top five processes annotated to cluster 5. Detailed microarray analysis of the fibroblast genes indicated that the cell population of the third passage exhibited the highest number of upregulated genes involved in the cell cycle and cell proliferation. In all cases, the results of qRT-PCR confirmed the differences in expression of the selected mRNAs between fibroblasts from the primary culture (C0) and from the first (C1), second (C2), and third (C3) cell passage. Our results thus suggest that these cells might be useful for increasing fibroblast numbers after reimplantation into a recipient's skin, and the method used in this study seems to be an excellent tool for autologous transplantation allowing the rejuvenation of aging skin.

Keywords: autologous fibroblasts; cell cycle genes; proliferation status; qRT-PCR; reimplantation; transcriptome profile.

Background: Mycosis fungoides (MF) is a primary cutaneous T-cell lymphoma (CTCL) that transforms from mature, skin-homing T cells and progresses during the early stages in the skin. The role of the skin microenvironment in MF development is unclear, but recent findings in a variety of cancers have highlighted the role of stromal fibroblasts in promoting or inhibiting tumorigenesis. Stromal fibroblasts are an important part of the cutaneous tumor microenvironment (TME) in MF. Here we describe studies into the interaction of TME-fibroblasts and malignant T cells to gain insight into their role in CTCL.

Methods: Skin from normal (n = 3) and MF patients (n = 3) were analyzed for FAPα by immunohistochemistry. MyLa is a CTCL cell line that retains expression of biomarkers TWIST1 and TOX that are frequently detected in CTCL patients. MyLa cells were cultured in the presence or absence of normal or MF skin derived fibroblasts for 5 days, trypsinized to detached MyL a cells, and gene expression analyzed by RT-PCR for MF biomarkers (TWIST1 and TOX), Th1 markers (IFNG, TBX21), Th2 markers (GATA3, IL16), and proliferation marker (MKI67). Purified fibroblasts were assayed for VIM and ACTA2 gene expression. Cellular senescence assay was performed to assess senescence.

Results: MF skin fibroblast showed increased expression of FAP-α with increasing stage compared to normal. Normal fibroblasts co-cultured with MyLa cells suppressed expression of TWIST1 (p < 0.0006), and TOX (p < 0.03), GATA3 (p < 0.02) and IL16 (p < 0.03), and increased expression of IFNG (p < 0.03) and TBX21 (p < 0.03) in MyLa cells. In contrast, MyLa cells cultured with MF fibroblasts retained high expression of TWIST1, TOX and GATA3. MF fibroblasts co-culture with MyLa cells increased expression of IL16 (p < 0.01) and IL4 (p < 0.02), and suppressed IFNG and TBX21 in MyLa cells. Furthermore, expression of MKI67 in MyLa cells was suppressed by normal fibroblasts compared to MF fibroblasts.

Conclusion: Skin fibroblasts represent important components of the TME in MF. In co-culture model, normal and MF fibroblasts have differential influence on T-cell phenotype in modulating expression of Th1 cytokine and CTCL biomarker genes to reveal distinct roles with implications in MF progression.

Keywords: Biomarkers; Cutaneous T-cell lymphoma; Fibroblasts; Mycosis fungoides; Tumor microenvironment.

Background: Proximal tubule cells dominate the kidney parenchyma numerically, although less abundant cell types of the distal nephron have disproportionate roles in water and electrolyte balance.

Methods: Coupling of a FACS-based enrichment protocol with single-cell RNA-seq profiled the transcriptomes of 9099 cells from the thick ascending limb (CTAL)/distal convoluted tubule (DCT) region of the mouse nephron.

Results: Unsupervised clustering revealed Slc12a3 ⁺/Pvalb ⁺ and Slc12a3 ⁺/Pvalb ^- cells, identified as DCT1 and DCT2 cells, respectively. DCT1 cells appear to be heterogeneous, with orthogonally variable expression of Slc8a1, Calb1, and Ckb. An additional DCT1 subcluster showed marked enrichment of cell cycle-/cell proliferation-associated mRNAs (e.g., Mki67, Stmn1, and Top2a), which fit with the known plasticity of DCT cells. No DCT2-specific transcripts were found. DCT2 cells contrast with DCT1 cells by expression of epithelial sodium channel β- and γ-subunits and much stronger expression of transcripts associated with calcium transport (Trpv5, Calb1, S100g, and Slc8a1). Additionally, scRNA-seq identified three distinct CTAL (Slc12a1 ⁺) cell subtypes. One of these expressed Nos1 and Avpr1a, consistent with macula densa cells. The other two CTAL clusters were distinguished by Cldn10 and Ptger3 in one and Cldn16 and Foxq1 in the other. These two CTAL cell types were also distinguished by expression of alternative Iroquois homeobox transcription factors, with Irx1 and Irx2 in the Cldn10 ⁺ CTAL cells and Irx3 in the Cldn16 ⁺ CTAL cells.

Conclusions: Single-cell transcriptomics revealed unexpected diversity among the cells of the distal nephron in mouse. Web-based data resources are provided for the single-cell data.

Keywords: distal convoluted tubule; macula densa; scRNA-seq; thick ascending limb.

Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); however, the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood. We previously studied the role of FOXL2^C134W, its partner SMAD3 and its antagonist FOXO1 in cellular models of aGCT.

Methods: In this work, seeking more comprehensive profiling of FOXL2^C134W transcriptomic effects, we performed an RNA-seq analysis comparing the effect of FOXL2^WT/SMAD3 and FOXL2^C134W/SMAD3 overexpression in an established human GC line (HGrC1), which is not luteinized, and bears normal alleles of FOXL2.

Results: Our data shows that FOXL2^C134W/SMAD3 overexpression alters the expression of 717 genes. These genes include known and novel FOXL2 targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and are enriched for neoplastic pathways (Proteoglycans in Cancer, Chromatin remodeling, Apoptosis, Tissue Morphogenesis, Tyrosine Kinase Receptors). We additionally expressed the FOXL2 antagonistic Forkhead protein, FOXO1. Surprisingly, overexpression of FOXO1 mitigated 40% of the altered genome-wide effects specifically related to FOXL2^C134W, suggesting it can be a new target for aGCT treatment.

Conclusions: Our transcriptomic data provide novel insights into potential genes (FOXO1 regulated) that could be used as biomarkers of efficacy in aGCT patients.

Keywords: Adult granulosa cell tumor; FOXL2; FOXO1; HGrC1; RNA-seq; SMAD3.

Background: Orofacial clefts (OFCs) are congenital malformations of the face and palate, with an incidence of 1 per 700 live births. Clubfoot or congenital talipes equinovarus (CTEV) is a three-dimensional abnormality of the leg, ankle, and feet that leads to the anomalous positioning of foot and ankle joints and has an incidence of 1 per 1000 live births. OFCs and CTEV may occur together or separately in certain genetic syndromes in addition to other congenital abnormalities. Here, we sought to decipher the genetic etiology of OFC and CTEV that occurred together in six probands.

Methods: At the time of recruitment, the most clinically obvious congenital anomalies in these individuals were the OFC and CTEV. We carried out whole-exome sequencing (WES) on DNA samples from probands and available parents employing the Agilent SureSelect XT kit and Illumina HiSeq2500 platform, followed by bioinformatics analyses. WES variants were validated by clinical Sanger Sequencing.

Results: Of the six probands, we observed probable pathogenic genetic variants in four. In three probands with probable pathogenic genetic variants, each individual had variants in three different genes, whereas one proband had probable pathogenic variant in just one gene. In one proband, we observed variants in DIS3L2, a gene associated with Perlman syndrome. A second proband had variants in EPG5 (associated with Vici Syndrome), BARX1 and MKI67, while another proband had potentially etiologic variants in FRAS1 (associated with Fraser Syndrome 1), TCOF1 (associated with Treacher Collins Syndrome 1) and MKI67. The last proband had variants in FRAS1, PRDM16 (associated with Cardiomyopathy, dilated, 1LL/Left ventricular noncompaction 8) and CHD7 (associated with CHARGE syndrome/Hypogonadotropic hypogonadism 5 with or without anosmia).

Conclusion: Our results suggest that clubfoot and OFCs are two congenital abnormalities that can co-occur in certain individuals with varying genetic causes and expressivity, warranting the need for deep phenotyping.

Keywords: clubfoot; genetic syndromes; orofacial clefts; whole-exome sequencing.

Tumor infiltrating immune cells plays a critical role in cancer progression. Apoptosis is an autonomous cell death that counteracts tumor growth. To this end, we hypothesized that increased apoptosis in breast cancer is associated with immune cell killing. Apoptosis score of MSigDB Hallmark collection was used to analyze METABRIC cohort (n=1904) and TCGA (n=1069) as validation cohort. High apoptosis tumors enriched cancer promoting signaling pathways; hypoxia, KRAS, TGF-β, PI3K signaling, and was associated with low MKI67 expression and less cell proliferation gene sets, less homologous recombination defects, and less altered fraction. High apoptosis tumors also enriched angiogenesis and high infiltration of vascular endothelial cells, pericytes and stromal cells and significantly enriched inflammation and immune response-related gene sets and high infiltration of CD8, CD4 memory, dendritic cells, M1 and M2 macrophages and significant elevation of cytolytic activity and immune checkpoint molecules, consistently in both cohorts. In conclusion, breast cancer patients with high apoptosis are associated with angiogenesis, immune response, high immune cell infiltration and cytolytic activity. To the best of our knowledge, this is the first study to utilize in silico translational approach to demonstrate the clinical relevance of apoptosis in breast cancer patients in large cohorts.

Keywords: Apoptosis; breast cancer; cytolytic activity; gene set; immune reaction; inflammatory response; tumor immune microenvironment.

Neoadjuvant Chemotherapy (NAC) is not frequently used in ER-positive/HER2-negative breast cancer (BC) because around 10% patients achieve pathological complete response (pCR). Since NAC can result in cancer downstaging both in the breast and axilla and prevent a morbid surgery, thus a score to predict pCR in this population will be crucial to identify patients who can benefit from this approach. A total of 4038 patients from cohorts; GSE25066, GSE20194, Hess, GSE20181, TCGA-BRCA and METBRIC were analyzed. The score was generated by the 5 most highly expressed genes in the Hallmark E2F targets gene set amongst patients in the GSE25066 cohort with ER-positive/HER2-negative BC who achieved pCR. The area under the curve was significantly higher in the score than that for the E2F targets score. High score ER-positive/HER2-negative BCs were significantly associated with higher Nottingham pathological grade, AJCC cancer stage, MKI67 expression levels, intratumor heterogeneity, homologous recombination defects, mutation burden, neoantigen load, and infiltration of anti-cancer immune cells (CD4⁺, T helper type1, plasmacytoid dendritic cells, M1 macrophages). They also expressed lower abundance of stromal cells including fibroblasts, lymphatic endothelial cells, pericytes and adipocytes consistently in GSE25066, TCGA and METABRIC cohorts. All cell proliferation-related gene sets, G2M checkpoint, E2F targets, MYC targets v1 and v2, Mitotic Spindle, were strongly enriched in high score BCs consistently in 3 cohorts. The gene score was significantly associated with high pCR rate consistently in the GSE25066 (38%, P < 0.001), GSE20194 (16%, P = 0.006), and Hess cohort (23%, P = 0.037). In conclusion, the 5-gene score reflects cancer cell proliferation and immune cell infiltration, and predicts pCR after NAC in ER-positive/HER2-negative breast cancer.

Keywords: 5-gene; ER-positive/HER2-negative breast cancer; neoadjuvant chemotherapy; predictive biomarker; tumor immune microenvironment.

Background and objectives: Receptor-interacting serine/threonine-protein kinase-2 (RIPK2) is an important mediator in different pathways in the immune and inflammatory response system. RIPK2 was also shown to play different roles in different cancer types; however, in colorectal cancer (CRC), its role is not well established. This study aims at identifying the role of RIPK2 in CRC progression and survival. Materials and methods: Data of patients and mRNA protein expression level of genes associated with CRC (RIPK2, tumor necrosis factor (TNF), TRAF1, TRAF7, KLF6, interlukin-6 (Il6), interlukin-8 (Il8), vascular-endothelial growth factor A (VEGFA), MKI67, TP53, nuclear factor-kappa B (NFKB), NFKB2, BCL2, XIAP, and RELA) were downloaded from the PrognoScan online public database. Patients were divided between low and high RIPK2 expression and different CRC characteristics were studied between the two groups. Survival curves were evaluated using a Kaplan-Meier estimator. The Pearson correlation was used to study the correlation between RIPK2 and the other factors. Statistical analysis was carried out using SPSS version 25.0. The Human Protein Atlas was also used for the relationship between RIPK2 expression in CRC tissues and survival. Differences were considered statistically significant at p < 0.05. Results: A total of 520 patients were downloaded from the PrognoScan database, and RIPK2 was found to correlate with MKI67, TRAF1, KLF6, TNF, Il6, Il8, VEGFA, NFKB2, BCL2, and RELA. High expression of RIPK2 was associated with high expression of VEGFA (p < 0.01) and increased mortality (p < 0.01). Conclusions: In this study, RIPK2 is shown to be a potential prognostic factor in CRC; however, more studies are needed to assess and verify its potential role as a prognostic marker and in targeted therapy.

Keywords: NFKB; RIPK2; colorectal cancer; inflammatory pathways.

Introduction: Molecular markers associated with breast cancer are assumed to be associated with outcome in non-muscle-invasive bladder cancer (NMIBC).

Materials and methods: We retrospectively investigated the association of the mRNA expression of estrogen receptor 1 (ESR1) and 2 (ESR2), progesterone receptor (PGR), MKI67, and HER2 (ERBB2) with recurrence-free (RFS), cancer-specific (CSS), and overall survival (OS) in 80 patients with stage T1 NMIBC.

Results: High expression of ESR2 (P = 0.003), ERBB2 (P < 0.001), and MKI67 (P = 0.029) was associated with shorter RFS. Only high ERBB2 was an independent prognostic factor for reduced RFS (HR = 2.98; P = 0.009). When sub stratifying the cohort, high ESR2 was associated with reduced RFS (P < 0.001), CSS (P = 0.037) and OS (P = 0.006) in patients without instillation therapy. High ESR2 was associated with reduced CSS (P = 0.018) and OS (P = 0.029) in females and with shorter RFS in both sexes (males: P = 0.035; females: P = 0.010). Patients with high ERBB2 showed reduced CSS (P = 0.011) and OS (P = 0.042) in females and reduced CSS (P = 0.012) in those without instillation, while RFS was significantly reduced irrespective of sex or instillation.

Conclusion: High mRNA expression of ERBB2 is an independent predictor of reduced RFS in patients with stage T1 NMIBC. High ERBB2 and ESR2 are associated with reduced outcomes, especially in females and patients without instillation therapy.

Keywords: ERBB2; Estrogen receptor; HER2; Non-muscle-invasive bladder cancer; Urothelial carcinoma.

Background: The purpose of this study was to assess the concordance of real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection of ESR1, PGR, ERBB2, and MKi67 messenger RNA (mRNA) in breast cancer tissues with central immunohistochemistry (IHC) in women treated within the prospective, randomized Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 6.

Patients and methods: We evaluated ESR1, PGR, ERBB2, and MKi67 mRNA expression by Xpert® Breast Cancer STRAT4 (enables cartridge-based RT-qPCR detection of mRNA in formalin-fixed paraffin-embedded tissues) and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 protein expression by IHC [in situ hybridization (ISH) for HER2 IHC 2+] in 1115 surgical formalin-fixed paraffin-embedded specimens from patients of ABCSG Trial 6. Overall percent agreement (concordance), positive percent agreement (sensitivity), and negative percent agreement (specificity) between STRAT4 and IHC were determined for each marker. The primary objective of the study was concordance between STRAT4 mRNA measurements of ESR1, PGR, ERBB2, and MKi67 with central reference laboratory IHC (and ISH for HER2 IHC 2+ cases). Time to distant recurrence was analyzed by Cox models.

Results: All performance targets for ER, PR, and Ki67 were met. For HER2, the negative percent agreement target but not the positive percent agreement target was met. Concordance between STRAT4 and IHC was 98.9% for ER, 89.9% for PR, 98.2% for HER2, and 84.8% for Ki67 (excluding intermediate IHC 10%-20% staining). In univariable and multivariable Cox regression analyses, all four biomarkers tested by either STRAT4 RT-qPCR or by central IHC (ISH) had a comparable time to distant recurrence indicating similar prognostic value.

Conclusions: With the exception of HER2, we demonstrate high concordance between centrally assessed IHC and mRNA measurements of ER, PR, and Ki67 as well as a high correlation of the two methods with clinical outcome. Thus, mRNA-based assessment by STRAT4 is a promising new tool for diagnostic and therapeutic decisions in breast cancer.

Keywords: early breast cancer; immunohistochemistry; postmenopausal women.

Ethnopharmacological relevance: Coptis chinensis Franch (CC) and Zingiber officinale Roscoe (dried ginger; DG) are traditional Chinese medicines. CC can dry dampness, relieve fire and detoxify, and is used to treat gastritis, gastric ulcer, colitis. DG can warm spleen and stomach for dispelling cold, used for the treatment of spleen and stomach deficiency. Both CC and DG are widely used to treat gastrointestinal diseases. CC-DG herb medicine combination originates from Huanglian decoction and Pinellia xiexin decoction in "Shanghan Lun" to comfort the stomach and intestines. CC and DG are used for the treatment of nausea and choking diaphragm which highly associated with gastric cancer clinically in ancient time.

Aim of the study: This study aimed to investigate the effects and underlying molecular mechanisms of CC-DG combination on gastric cancer.

Materials and methods: The CC-DG extract was subjected to HPLC analysis. Viability (MTT) and cytotoxicity (CCK8) assays were performed using the SGC7901 and MFC cells. Cell cycle and apoptosis were measured by flow cytometry. The mRNA expression levels were measured by RT-PCR. In vivo anti-tumor activity of CC-DG was assessed in a tumor xenograft model.

Results: Twelve different proportions of CC-DG were tested for inhibitory effects on gastric cancer cells; CC-DG ratio 1:1 was found most effective. CC-DG administration significantly reduced the cell proliferation, migration, and colony formation, while increased cell apoptosis compared with the control group. CC-DG regulated differentially expressed genes in SGC7901 cells were subjected to pathway enrichment analysis. CC-DG significantly inhibited the cell glucose metabolism, downregulated the expression of LDHA and SLC2A1 genes, and changed the expression of other related genes including ME2, LDHD, LDHB, HIF1A, PKM, Pcx, and Got1. In addition, CC-DG suppressed tumorigenesis and inhibited MKI67 expression in the tumor xenograft model.

Conclusions: CC-DG inhibited the proliferation, migration, invasion of SGC7901/MFC gastric cells, and in turn, suppressed tumorigenesis by regulating glucose metabolism through regulation of LDHA and SLC2A1 genes.

Keywords: Apoptosis; Coptis chinensis-Dried ginger herb combination; Gastric cancer; Glucose metabolism.

Breast cancer clinical treatment selection is based on the immunohistochemical determination of four protein biomarkers: ESR1, PGR, HER2, and MKI67. Our aim was to correlate immunohistochemical results to proteome-level technologies in measuring the expression of these markers. We also aimed to integrate available proteome-level breast cancer datasets to identify and validate new prognostic biomarker candidates. We searched studies involving breast cancer patient cohorts with published survival and proteomic information. Immunohistochemistry and proteomic technologies were compared using the Mann-Whitney test. Receiver operating characteristics (ROC) curves were generated to validate discriminative power. Cox regression and Kaplan-Meier survival analysis were calculated to assess prognostic power. False Discovery Rate was computed to correct for multiple hypothesis testing. We established a database integrating protein expression data and survival information from four independent cohorts for 1229 breast cancer patients. In all four studies combined, a total of 7342 unique proteins were identified, and 1417 of these were identified in at least three datasets. ESR1, PGR, and HER2 protein expression levels determined by RPPA or LC-MS/MS methods showed a significant correlation with the levels determined by immunohistochemistry (p < 0.0001). PGR and ESR1 levels showed a moderate correlation (correlation coefficient = 0.17, p = 0.0399). An additional panel of candidate proteins, including apoptosis-related proteins (BCL2,), adhesion markers (CDH1, CLDN3, CLDN7) and basal markers (cytokeratins), were validated as prognostic biomarkers. Finally, we expanded our previously established web tool designed to validate survival-associated biomarkers by including the proteomic datasets analyzed in this study ( https://kmplot.com/ ). In summary, large proteomic studies now provide sufficient data enabling the validation and ranking of potential protein biomarkers.

Some cannabinoids showed anti-inflammatory and antifibrotic activities. EHP-101 is an oral lipidic formulation of the novel non-psychotropic cannabidiol aminoquinone VCE-004.8, which showed antifibrotic activity in murine models of systemic sclerosis induced by bleomycin. We herein examined the effect of EHP-101 on cardiac and other organ fibrosis in a mouse model induced by Angiotensin II. VCE-004.8 inhibited TGFβ- and Ang II-induced myofibroblast differentiation in cardiac fibroblasts detected by α-SMA expression. VCE-004.8 also inhibited Ang II-induced ERK 1 + 2 phosphorylation, NFAT activation and mRNA expression of IL1β, IL6, Col1A2 and CCL2 in cardiac fibroblasts. Mice infused with Ang II resulted in collagen accumulation in left ventricle, aortic, dermal, renal and pulmonary tissues; oral administration of EHP-101, Ajulemic acid and Losartan improved these phenotypes. In myocardial tissue, Ang II induced infiltration of T cells and macrophages together with the accumulation of collagen and Tenascin C; those were all reduced by either EHP-101 or Losartan treatment. Cardiac tissue RNA-Seq analyses revealed a similar transcriptomic signature for both treatments for inflammatory and fibrotic pathways. However, the gene set enrichment analysis comparing data from EHP-101 vs Losartan showed specific hallmarks modified only by EHP-101. Specifically, EHP-101 inhibited the expression of genes such as CDK1, TOP2A and MKi67 that are regulated to the E2 factor family of transcription factors. This study suggests that the oral administration of EHP-101 prevents and inhibits cardiac inflammation and fibrosis. Furthermore, EHP-101 inhibits renal, pulmonary and dermal fibrosis. EHP-101 could offer new opportunities in the treatment of cardiac fibrosis and other fibrotic diseases.

Keywords: Angiotensin II; Cannabinoids; Cardiac Fibrosis; E2F; EHP-101.

Background: Targeted programmed cell death protein 1 (PD-1) therapy could effectively improve the long-term prognosis of patients with non-small cell lung cancer (NSCLC). The role of PD-1 targets in the progression of NSCLC has not been fully revealed.

Methods: The differentially expressed genes (DEGs) in patients' blood after NSCLC treatment with PD-1 blocker nivolumab in the GSE141479 dataset were analyzed by GEO2R and identified in the TCGA database. The mechanism of action involved in the PD-1 target molecules via the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein-protein interaction (PPI) network shows the relationship between PD-1 target molecules. The factors affecting the prognosis of NSCLC patients were identified via the COX regression analysis and survival analysis to build the risk model and nomogram.

Results: There were 64 DEGs in patients' blood after nivolumab treatment and 48 DEGs in NSCLC tissues. The PD-1 target molecules involved cell proliferation, DNA replication, cell cycle, lung cancer, and other cellular processes. The prognostic factors CCNA2, CHEK1, DLGAP5, E2F8, FOXM1, HIST1H2BH, HJURP, MKI67, PLK1, TPX2, and TYMS, and the independent factors HIST1H2BH and PLK1, influenced the prognosis of NSCLC patients. HIST1H2BH and PLK1 were overexpressed in LUAD and LUSC tissues. The elevated expression levels of HIST1H2BH and PLK1 were related to the overall survival (OS) and the progression-free survival of NSCLC patients. High-risk NSCLC patients had a poor prognosis and were an independent factor influencing the poor prognosis of NSCLC patients. The high-risk model group was enriched with signaling mechanisms such as cell cycle, DNA replication, and homologous recombination.

Conclusions: The risk model based on PD-1 target molecules was helpful to assess the prognosis of NSCLC patients. HIST1H2BH and PLK1 might become prognostic biomarkers of NSCLC patients.

Keywords: Programmed cell death protein 1 (PD-1); nivolumab; non-small cell lung cancer (NSCLC).

Early researches have revealed that circular RNA (circRNA) had the potential of biomarkers and could affect tumor progression through regulatory networks. However, few research focused on the function of circRNA in lung adenocarcinoma and the regulation mechanism of competitive endogenous RNA. In present study, through differential expression analysis, 10 circRNAs, 98 miRNAs(microRNA) and 2497 mRNAs were screened. Based on the 10 circRNAs and related databases, a competitive endogenous RNA regulatory network (ceRNA network) containing 7 circRNAs, 13 miRNAs and 147 mRNAs was constructed. KEGG and GO analysis suggested that 147 mRNAs were obviously enriched in biological pathway related to LUAD. By constructing a PPI network, 12 hub genes were identified by MCODE. The result of survival analysis showed that 10 hub genes (BIRC5, MKI67, CENPF, RRM2, BUB1, MELK, CEP55, CDK1, NEK2, TOP2A) were significantly related to the survival of LUAD. We randomly divided 483 clinical data into two parts: train set and validation set. The train set was used for Cox regression analysis, 3 prognostic factors (stage, T, CDK1) were screened. The nomogram model was constructed based on stage, T and CDK1. The model was evaluated by ROC curve, calibration chart, Kaplan-Meier (KM) curve and validation set data. The results indicated that the model has good accuracy. Our study elucidated the regulatory mechanism of circRNA in lung adenocarcinoma, and the nomogram model also provided insight for the clinical analysis of lung adenocarcinoma.

Keywords: bioinformatics; circular RNA; competitive endogenous network; lung adenocarcinoma; nomogram model.

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker β-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.

Keywords: ROCK inhibitor; Y-27632; mesenchymal stem cell-derived conditioned medium; rabbit corneal endothelial cells; senescence.