BACKGROUND

Prediction of response to preoperative breast cancer chemotherapy may offer a substantial optimization of medical management of this disease. The most efficient prediction would be done a priori, before the start of chemotherapy and based on the biological features of patient and tumor. Numerous markers have been proposed but none of them has been applied as a routine. The role of MKI67 and HSP90 expression has been recently suggested to predict treatment sensitivity in HER2-positive breast cancer. The aim of this study was to validate the utility of proliferation based markers (MKI67 and CDK1) and heat shock proteins (namely HSP90) to predict response to chemotherapy in cohort of breast cancer patients treated preoperatively.

METHODS

Ninety-three patients with breast cancer, all females, mean age 42.2 years, among them 32% T1-T2 patients, 49% T3 patients and 13% with T4 tumor stage, 27% N0, 42% N1, 16% N2, 15% N3 were subjected to initial chemotherapy. The majority of patients (86%) received anthracycline and taxane chemotherapy. Among the patients there were 9 individuals with metastatic disease (M1) at initial presentation, and 11 patients were not treated surgically after initial chemotherapy (no sufficient disease response). From 82 patients operated on, 20 patients (24%) showed pathological complete response (pCR), while in 62 patients there was no pCR. 42% of patients were hormone-sensitive HER2-negative, 20% hormone-sensitive HER2-positive, 9% only HER-positive and 29% with triple negative breast cancer. Four gene transcripts (MKI67, cyclin-dependent kinase 1 [CDK1], heat shock proteins HSP90AA1 and HSP- 90AB1) were analyzed in total RNA isolated from single core obtained during preoperative core needle biopsy by quantitative real-time PCR with fluorescent probes (Universal Probe Library, Roche). Results were normalized to the panel of reference genes.

RESULTS

There were no statistically significant differences in MKI67 and CDK1 expression between pCR and no pCR groups (p = 0.099 and 0.35, respectively), although the median expression of both genes was slightly higher in pCR group. In contrast, both HSP90AA1 and HSP90AB1 transcripts showed decreased expression in pCR group (medians 0.77 and 0.55) when compared to no p CR group (median 0.86 and 0.73), statistically significant for HSP90AA1 (p = 0.031) and of borderline significance for HSP90AB1 (p = 0.054). The most significant predictor of pCR was the ratio of CDK1 transcript to HSP90AA transcript. This ratio was significantly higher in CR group (median 0.99) than in no CR group (median 0.68, p = 0.0023), and showed a potential diagnostic utility (area under receiver operating characteristic [ROC] curve 0.72).

CONCLUSIONS

HSP90AA1 and AB1 genes exhibit low expression in breast cancers highly sensitive to chemotherapy and may indicate the patients with higher probability of pathological complete response. The ratio of HSP90AA1 to proliferation-related markers (CDK1 or MKI67) may be even better predictor of pCR chance, with higher expression of proliferation genes and lower stress response in patients sensitive to chemotherapy.

The presence of circulating tumor cells (CTCs), detected as a form of liquid biopsy is associated with poor survival in both early and metastatic breast cancer. Monitoring tumor biology based on intrinsic subtypes delivers treatment-relevant information on the heterogeneity or biomarker conversion between primary and metastatic tumors. This study aimed to correlate the change of the apoptotic and intact CTC counts with mRNA-assessed intrinsic subtype change. Thirty-four breast cancer patients with available triplets of primary tumors, distant metastasis biopsies and data on intact and apoptotic CTC dynamics were included in the analysis. The intrinsic subtype was determined per RT-qPCR quantification of the gene expression ESR1, PGR, ERBB2 and MKI67. Both luminal (p = 0.038) and triple negative (p = 0.035) patients showed a significant downregulation of apoptotic CTCs. Repeated biopsies of distant metastatic sites, as well as determining a potential shift of the intrinsic subtype, combined with data on intact and apoptotic CTC dynamics from liquid biopsies might help personalize systemic therapy and generate additional surrogate markers for successful systemic therapy.

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186-96. ©2016 AACR.

The efficacy of ERr 731(®), a commercially available extract isolated from Rheum rhaponticum, in terms of menopausal complaints like hot flushes, depression, anxiety and vaginal dryness has been proven in a two-year clinical study. Further a recent preclinical study excluded unwanted side effects on the endometrium by showing a lack of stimulation of proliferation marker genes by ERr 731(®) or its constituents in the 3-day uterotrophic assay. The present study aimed at further substantiating the safety of ERr 731(®) in terms of endometrial hyperplasia and at the same time test for potential estrogenic effects in the bone. Therefore, ovariectomized (ovx) rats were treated in a dietary long-term administration for 90 days. Hence, the modulation of proliferation in the uterus was investigated by examining the effects on the mRNA expression of proliferation marker genes (Mki67, Pcna), on the estrogen-responsive gene C3 and on the estrogen receptors ERα and ERβ. We additionally performed densitometry analysis of the proximal tibia metaphysis using peripheral computed tomography (pQCT) and quantified bone homeostasis markers in the serum to examine potential effects on the bone. In this study design, neither an uterotrophic response nor a modulation of proliferation marker genes on mRNA level has been observed as response to the long-term application of the rhapontic extract. Furthermore, no impact of the two administered ERr 731(®) doses on the E2 deprivation-induced bone loss has been evident at the end of the study. In conclusion, the observations from previous trials regarding the endometrial safety of ERr 731(®) have been supported by our experimental findings that exclude a stimulatory activity on proliferation in the uterus in a long-term administration in the young adult rat but no effect on the bone mineral density could be observed.

BACKGROUND

LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin.

OBJECTIVE

The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin.

METHODS

FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy.

RESULTS

LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration.

CONCLUSIONS

In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration.

Deer antlers are unique cranial appendages capable of regeneration and rapid growth. In addition, deer antlers have been widely used in traditional Chinese medicine to promote the function of the kidneys, reproductive system, bones and nervous system. It has been shown that water-soluble substances are the major bioactive components within the deer antlers. In this study, we prepared aqueous extracts from deer antlers during a rapid growth stage. We investigated the effects of antler extracts on primary chondrocytes by analyzing their protein expression patterns using isobaric tags for relative and absolute quantitation technology. We demonstrated that antler extracts promote chondrocyte proliferation and prevent chondrocyte differentiation and apoptosis by controlling multiple cellular processes involved in genomic stability, epigenetic alterations, ribosome biogenesis, protein synthesis and cytoskeletal reorganization. Antler extracts significantly increased the expression levels of proliferation markers Mki67 and Stmn1 and differentiation inhibitor Acp5 as well as cellular apoptosis inhibitors Ndufa4l2 and Rcn1. Thus, this study has greatly expanded our current knowledge of the molecular effects of antler extracts on chondrocytes. It has also shed new light on possible strategies to prevent damage to and to treat cartilage and its related diseases by using aqueous extracts from growing Sika deer antlers.

Notch signaling as a conserved cell fate regulator is involved in the regulation of cell quiescence, proliferation, differentiation and postnatal tissue regeneration. However, how Notch signaling regulates porcine satellite cells (PSCs) has not been elucidated. We stably transfected Notch1 intracellular domain (N1ICD) into PSCs to analyze the gene expression profile and miRNA-seq. The analysis of the gene expression profile identified 295 differentially-expressed genes (DEGs) in proliferating-N1ICD PSCs (P-N1ICD) and nine DEGs on differentiating-N1ICD PSCs (D-N1ICD), compared with that in control groups (P-Control and D-Control, respectively). Analyzing the underlying function of DEGs showed that most of the upregulated DEGs enriched in P-N1ICD PSCs are related to the cell cycle. Forty-four and 12 known differentially-expressed miRNAs (DEMs) were identified in the P-N1ICD PSCs and D-N1ICD PSCs group, respectively. Furthermore, we constructed the gene-miRNA network of the DEGs and DEMs. In P-N1ICD PSCs, miR-125a, miR-125b, miR-10a-5p, ssc-miR-214, miR-423 and miR-149 are downregulated hub miRNAs, whose corresponding hub genes are marker of proliferation Ki-67 (MKI67) and nuclear receptor binding SET domain protein 2 (WHSC1). By contrast, miR-27a, miR-146a-5p and miR-221-3p are upregulated hub miRNAs, whose hub genes are RUNX1 translocation partner 1 (RUNX1T1) and fibroblast growth factor 2 (FGF2). All the hub miRNAs and genes are associated with cell proliferation. Quantitative RT-PCR results are consistent with the gene expression profile and miRNA-seq results. The results of our study provide valuable information for understanding the molecular mechanisms underlying Notch signaling in PSCs and skeletal muscle development.

Hepatoblastoma (HB) is one of the most common liver malignancies in children, while the molecular basis of the disease is largely unknown. Therefore, this study aims to explore the key genes and molecular mechanisms of the pathogenesis of HB using a bioinformatics approach. The gene expression dataset GSE131329 was used to find differentially expressed genes (DEGs). Functional and enrichment analyses of the DEGs were performed by the EnrichR. Then, the protein-protein interaction (PPI) network of the up-regulated genes was constructed and visualized using STRING database and Cytoscape software, respectively. MCODE was used to detect the significant modules of the PPI network, and cytoHubba was utilized to rank the important nodes (genes) of the PPI modules. Overall, six ranking methods were employed and the results were validated by the Oncopression database. Moreover, the upstream regulatory network and the miRNA-target interactions of the up-regulated DEGs were analyzed by the X2K web and the miRTarBase respectively. A total of 594 DEGs, including 221 up- and 373 down-regulated genes, were obtained, which were enriched in different cellular and metabolic processes, human diseases, and cancer. Furthermore, 15 hub genes were screened, out of which, 11 were validated. Top 10 transcription factors, kinases, and miRNAs were also determined. To the best of our knowledge, the association of RACGAP1, MKI67, FOXM1, SIN3A, miR-193b, and miR-760 with HB was reported for the first time. Our findings may be used to shed light on the underlying mechanisms of HB and provide new insights for better prognosis and therapeutic strategies.

Keywords: Bioinformatic analysis; Biomarker; Gene regulatory network; Hepatoblastoma; Protein kinase; miRNAs.

Minichromosome maintenance complex component 6 (MCM6) is involved in tumorigenesis of hepatocellular carcinoma (HCC). Because its effect on different populations remains unclear, this study investigated the impact of MCM6 on HCC in Southern Chinese Zhuang population. In addition to assessing the global mRNA levels of MCM6 based on The Cancer Genome Atlas database (TCGA) and The Gene Expression Omnibus database (GEO), associations between MCM6 mRNA levels and clinicopathological features were analyzed. High MCM6 levels were associated with high alpha-fetoprotein (AFP) (>20 ng/mL in serum) (P < 0.0001) and advanced clinical stage (III + IV) (P < 0.001). Higher MCM6 was associated with poorer outcomes (P < 0.01) in these databases. Furthermore, the mRNA and protein expression of MCM6 in the Guangxi Zhuang population was detected by quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry (IHC). The results showed that MCM6 levels were up-regulated in the Zhuang population with HCC. Higher MCM6 protein levels were correlated with larger tumor size (>5 cm) (P = 0.038) and advanced clinical stage (III + IV) (p = 0.023). Bioinformatic enrichment analysis of MCM6 and its interacting proteins (CDT1,WEE1,TRIM28 and MKI67) suggested that in addition to being involved in the cell cycle process, these complexes could also be involved in protein binding, pre-replication complex assemble, and nucleus metabolism. Based on the protein-protein interaction (PPI) network with module screen, the interactions between MCM6 and its potential interacting proteins were further studied through protein docking with hot spot analysis. Additionally, the results of the algorithms combining the ROC of MCM6 and its interacting proteins showed that combination biomarker analysis has better HCC diagnosis ability than the single MCM6 test. The combination of MCM6 and TRIM28 was more suitable for the Guangxi Zhuang population. Overall, our study suggests that MCM6 plays an important role in the growth of HCC. MCM6 could be an optimal biomarker for diagnosing HCC and a potential molecular target for HCC therapy in the Zhuang population.

OBJECTIVE

To investigate the role of forkhead box protein M1 (FOXM1) mRNA expression and its prognostic value in stage pT1 non-muscle-invasive bladder cancer (NMIBC).

METHODS

Clinical data and formalin-fixed paraffin-embedded tissues from transurethral resection of the bladder from patients with stage pT1 NMIBC, treated with an organ-preserving approach, were analysed retrospectively. Total RNA was isolated using commercial RNA extraction kits, and mRNA expression of FOXM1, MKI67, KRT20 and KRT5 was measured by single-step quantitative RT-PCR using RNA-specific TaqMan Assays. Statistical analysis was performed using Spearman's Rho, Wilcoxon or Kruskal-Wallis tests, Kaplan-Meier estimates of recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS) and Cox regression analysis.

RESULTS

Data from 296 patients (79.4% men, median age 72 years) were available for the final evaluation. Spearman correlation analysis showed that mRNA expression of FOXM1 was significantly correlated with MKI67 (ρ: 0.6530, P < 0.001) and with the luminal subtype, reflected by the positive correlation with KRT20 (ρ: 0.2113, P < 0.001). Furthermore, there was also a strong correlation of FOXM1 expression with adverse clinical and pathological variables, such as concomitant carcinoma in situ (P = 0.05), multifocal tumours (P = 0.005) and World Health Organization 1973 grade 3 disease (P < 0.001). Kaplan-Meier analysis showed overexpression of FOMX1 to be associated with worse PFS (P = 0.028) and worse CSS (P = 0.015). FOXM1 overexpression was also shown to be a predictive risk factor for CSS (hazard ratio 1.61 [1.13-2.34], L-R chi-squared: 7.19, P = 0.007). FOXM1 overexpression identified a subgroup of patients within the luminal subtype with worse RFS (P = 0.017), PFS (P < 0.001) and CSS (P = 0.015). Patients with low FOXM1 expression had better outcomes, irrespective of instillation therapy, whereas patients with high FOXM1 expression benefitted from intravesical chemotherapy with mitomycin C.

CONCLUSIONS

High FOXM1 expression was associated with adverse clinical and pathological features and worse outcomes, and predicted response to intravesical instillation therapy in patients with stage pT1 NMIBC.

Forkhead box M1 (FOXM1) is a late cell cycle gene that plays a crucial role in carcinogenesis and chemotherapeutic drug resistance. In this study, the impact of FOXM1 expression on patient outcome was investigated for the first time in formalin fixed and paraffin embedded (FFPE) samples of chemotherapy naïve muscle-invasive bladder cancer (MIBC) patients. Expression analyses were performed on the Mannheim cohort (n=84) and validated on the independent Chungbuk cohort (n=61). In a Cox' proportional hazards model, a distinct FOXM1 expression cut-off dividing both cohorts in a 'high-risk' and 'low-risk' group has been determined. Multivariate analyses showed that FOXM1 is an independent risk factor for outcome prediction superior to the TNM system. The FOXM1 'high-risk' group had a 4- to 7-fold increased risk of death (p<0.03) and presented further an overexpression of MKI67. Recent studies showed that MIBCs can be subclassified in breast cancer-like subtypes: basal, luminal and p53-like. Here we demonstrated that FOXM1 was differentially expressed between MIBC subtypes concordant to its subtype specific expression in breast cancer. Since the proto-oncogene FOXM1 is known to play an important role in cisplatin resistance and to be a promising drug target, this study supports FOXM1 as a crucial biomarker in the personalization of MIBC therapy and urges prospective translational studies.

Extracellular fragments derived from plasma membrane receptors can play relevant roles in the development/progression of tumor pathologies, thereby offering novel diagnostic or therapeutic opportunities. The truncated variant of somatostatin receptor subtype-5, SST5TMD4, is an aberrantly spliced receptor with 4 transmembrane domains, highly overexpressed in several tumor types, whose C-terminal tail is exposed towards the extracellular matrix, and could therefore be the substrate for proteolytic enzymes. In silico analysis implemented herein predicted 2 possible cleavage sites for metalloproteases MMP2, 9, 14, and 16 in its sequence, which could generate 3 releasable peptides. Of note, expression of those MMPs was directly correlated with SST5TMD4 in several cancer-derived cell lines (ie neuroendocrine tumors and prostate, breast, and liver cancers). Moreover, incubation with SST5TMD4-derived peptides enhanced malignancy features in all cancer cell types tested (ie proliferation, migration, etc.) and blunted the antiproliferative response to somatostatin in QGP-1 cells, acting probably through PI3K/AKT and/or MEK/ERK signaling pathways and the modulation of key cancer-associated genes (eg MMPs, MKI67, ACTR2/3, CD24/44). These results suggest that SST5TMD4-derived peptides could contribute to the strong oncogenic role of SST5TMD4 observed in multiple tumor pathologies, and, therefore, represent potential candidates to identify novel diagnostic, prognostic, or therapeutic targets in cancer.

Lung cancer is the leading cause of cancer-related mortality worldwide. Despite progress in the treatment of non-small-cell lung cancer, there are limited treatment options for lung squamous cell carcinoma (LUSC), compared with lung adenocarcinoma. The present study investigated the disease mechanism of LUSC in order to identify key candidate genes for diagnosis and therapy. A total of three gene expression profiles (GSE19188, GSE21933 and GSE74706) were analyzed using GEO2R to identify common differentially expressed genes (DEGs). The DEGs were then investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. A protein-protein interaction (PPI) network was constructed via the Search Tool for the Retrieval of Interacting Genes/Proteins, and visualized using Cytoscape software. The expression levels of the hub genes identified using CytoHubba were validated using the University of California, Santa Cruz (UCSC) database and the Human Protein Atlas. A Kaplan-Meier curve and Gene Expression Profiling Interactive Analysis were then employed to evaluate the associated prognosis and clinical pathological stage of the hub genes. Furthermore, non-coding RNA regulatory networks were constructed using the Gene-Cloud Biotechnology information website. A total of 359 common DEGs (155 upregulated and 204 downregulated) were identified, which were predominantly enriched in 'mitotic nuclear division', 'cell division', 'cell cycle' and 'p53 signaling pathway'. The PPI network consisted of 257 nodes and 2,772 edges, and the most significant module consisted of 66 upregulated genes. A total of 19 hub genes exhibited elevated RNA levels, and 10 hub genes had elevated protein levels compared with normal lung tissues. The upregulation of five hub genes (CCNB1, CEP55, FOXM1, MKI67 and TYMS; defined in Table I) were significantly associated with poor overall survival and unfavorable clinical pathological stages. Various ncRNAs, such as C1orf220, LINC01561 and MGC39584, may also play important roles in hub-gene regulation. In conclusion, the present study provides further understanding of the pathogenesis of LUSC, and reveals CCNB1, CEP55, FOXM1, MKI67 and TYMS as potential biomarkers or therapeutic targets.

Most breast cancer (BC) patients succumb to metastatic disease. MiR-34a is a well-known tumor suppressive microRNA which exerts its anti-cancer functions by playing a role in p53, apoptosis induction, and epithelial-mesenchymal transition (EMT) suppression. Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) cohorts were used to test our hypothesis that miR-34a high BCs translate to less aggressive cancer biology and better survival in large cohorts. There was no association between miR-34a expression levels and clinicopathological features of BC patients except for HER2 positivity. MiR-34a high expressing tumors were associated with lower Nottingham pathological grades and lower MKI67 expression. In agreement, high miR-34a tumors demonstrated lower GSVA scores of cell cycle and cell proliferation-related gene sets. High miR-34a tumors enriched the p53 pathway and apoptosis gene sets. Unexpectedly, high miR-34a tumors also associated with elevated EMT pathway score and ZEB1 and two expressions. MiR-34a expression did not associate with any distant metastasis. Further, high miR-34a tumors did not associate with better survival compared with miR-34a low tumors. In conclusion, the clinical relevance of miR-34a high expressing tumors was associated with suppressed cell proliferation, enhanced p53 pathway and apoptosis, but enhanced EMT and these findings did not reflect better survival outcomes in large BC patient cohorts.

Endometrial cancer (EC) is one of the most common female cancers. One of the key processes involved in EC development is uncontrolled proliferation stimulated by local factors such as angiotensin. The aim of the present study was to evaluate the influence of angiotensin II (Ang II) on human EC cells. Biological assays and gene expression analysis were performed on three cell lines: ISH, MFE-296 and MFE-280. Our results indicated that at the beginning of cancerogenesis Ang II induced abnormal proliferation at lower doses. We also showed that dose-dependent induction of proliferation was connected with changes in the expression of MKI67, CCND1 and CCNE1 genes in well- and poorly differentiated cancer cells. After Ang II treatment, poorly differentiated endometrial cancer cell line acquired a mesenchymal phenotype, which was characterized by induced expression of EMT-related genes (VIM, CD44, SNAI1, ZEB1 and ZEB2). Our study revealed that Ang II influences EC cells in terms of cancer-related processes, and is responsible for increased proliferation, reduction in apoptosis, increased mobility and modulation of adhesion potential. Its effect and effectiveness appear to be highly connected with the differentiation status of the cancerous cells, as Ang II appears to play a crucial role in the early and late stages of malignant transformation.

Preeclampsia (PE) is a life-threatening complication of human pregnancy with no effective treatment other than premature delivery. It is hallmarked by systemic endothelial injury/dysfunction which is believed to be caused by abnormal levels/types of placenta-derived factors that are circulating in the maternal blood. Emerging evidence suggests that endothelial repair is also dysregulated in PE, as circulating endothelial progenitor cells (EPCs) critical for endothelial regeneration are reduced in number and functionality. However, the underlying mechanisms are poorly understood. HtrA4 is a placenta-specific protease that is secreted into the circulation and significantly elevated in early-onset PE. Here we investigated the impact of HtrA4 on endothelial proliferation and repair. We demonstrated that high levels of HtrA4 halted endothelial cell proliferation and significantly down-regulated a number of genes that are critical for cell cycle progression, including CDKN3, BIRC5, CDK1 and MKI67. Furthermore, HtrA4 significantly inhibited the proliferation of primary EPCs isolated from term human umbilical cord blood and impeded their differentiation into mature endothelial cells. Our data thus suggests that elevated levels of HtrA4 in the early-onset PE circulation may impair endothelial cell repair, not only by halting endothelial cell proliferation, but also by inhibiting the proliferation and differentiation of circulating EPCs.

Pancreatic cancer is highly mortal due to uncontrolled cell proliferation. The G2M checkpoint pathway is an essential part of the cell cycle. We hypothesized that a high G2M pathway score is associated with cell proliferation and worse survival in pancreatic cancer patients. Gene set variation analysis using the Hallmark G2M checkpoint gene set was used as a score to analyze a total of 390 human pancreatic cancer patients from 3 cohorts (TCGA, GSE62452, GSE57495). High G2M score tumors enriched other cell proliferation genes sets as well as MKI67 expression, pathological grade, and proliferation score. Independent of other prognostic factors, G2M score was predictive of disease-specific survival in pancreatic cancer. High G2M tumor was associated with high mutation rate of KRAS and TP53 and significantly enriched these pathway gene sets, as well as high infiltration of Th2 cells. High G2M score consistently associated with worse overall survival in 3 cohorts, particularly in R1/2 resection, but not in R0. High G2M tumor in R1/2 highly enriched metabolic and cellular components' gene sets compared to R0. To our knowledge, this is the first study to use gene set variation analysis as a score to examine the clinical relevancy of the G2M pathway in pancreatic cancer.

Keywords: biomarker; cell cycle; gene set; pancreatic cancer; pathway analysis; tumor gene expression.

Hepatocyte growth factor (HGF) and its receptor MET have been implicated in uterine development, pregnancy, and endometrial disorders, such as endometriosis and carcinoma. In vitro studies have shown that HGF acts as a mitogen, motogen, and morphogen on endometrial epithelial cells. However, the expression and regulation of HGF and MET in the uteri of different species remain obscure. The present study aimed to investigate the changes of HGF, MET, and HGF activator (HGFA) expression in the uterine endometrium during the estrous cycle in mice and to explore estrogen and progesterone regulation of their expression. MKI67 immunostaining was conducted to examine the association between HGF/MET expression and endometrial cell proliferation. Endometrial epithelial and stromal cells both expressed HGF, HGFA, and MET, but the cell type-specific patterns changed during the cycle. Estrogen and progesterone differentially regulated HGF, MET, and HGFA expression. Progesterone up-regulated their expression in the stroma and down-regulated their expression in the luminal epithelium, whereas 17-beta-estradiol down-regulated their expression in the glandular epithelium. The pattern of HGF/MET overall correlated with that of MKI67. In conclusion, HGF, HGFA, and MET expression in mouse uterus changes during the estrous cycle in a stage-, cell type-, and compartment-specific manner under the influence of estrogen and progesterone. HGF likely plays a role in cyclic endometrial remodeling, such as cell proliferation via autocrine/paracrine mechanisms in mouse uterus.

Dysregulation of the cell cycle has been observed and implicated as an etiologic factor in a range of human malignancies, but remains relatively unstudied in neuroendocrine tumors (NETs). We evaluated expression of key proteins involved in cell cycle regulation in a large cohort of NETs.

We evaluated immunohistochemical expression of CDKN1B, CDKN1A, CDKN2A, CDK2, CDK4, CDK6, cyclin D1, cyclin E1, and phosphorylated retinoblastoma protein (phospho-RB1) in a cohort of 267 patients with NETs. We then explored associations between cell cycle protein expression, mutational status, histologic features, and overall survival.

We found that high expression of CDK4, CDK6, CCND1, and phospho-RB1 was associated with higher proliferative index, as defined by MKI67. We additionally observed a trend toward shorter overall survival associated with low expression of CDKN1B. This association seemed strongest in SINETs (multivariate hazards ratio, 2.04; 95% confidence interval, 1.06-3.93; P = 0.03). We found no clear association between CDKN1B mutation and protein expression.

Our results suggest that dysregulation and activation of the CDK4/CDK6-CCND1-phospho-RB1 axis is associated with higher proliferative index in NETs. Investigation of the therapeutic potential of CDK4/CDK6 inhibitors in higher grade NETs is warranted.

Anaplastic oligodendroglioma (AO), IDH-mutant and 1p/19q codeleted (IDHmut+/1p19qcodel), is a high-grade glioma with only limited prognostic markers. The primary objective of this study was to evaluate, by immunohistochemistry, the prognostic value of two proliferation markers, MCM6 and Ki-67, in a large series of IDHmut+/1p19qcodel AO included in the POLA ("Prise en charge des Oligodendrogliomes Anaplasiques") French national multicenter network. We additionally examined the transcriptome obtained from this series to understand the functional pathways dysregulated with the mRNA overexpression of these two markers. The labelling indices (LI) of MCM6 and Ki-67 were obtained via computer-assisted color image analyses on immunostained AO tissues of the cohort (n=220). Furthermore, a subgroup of AO (n=68/220) was used to perform transcriptomic analyses. A high LI of either MCM6 (≥50%) or Ki-67 (≥ 15%) correlated with shorter overall survival, both in univariate (P=0.013 and P=0.004, respectively) and multivariate analyses (P=0.027; multivariate Cox model including age, mitotic index, MCM6 and Ki-67). MCM6 and Ki-67 LI also correlated with overall survival in an additional retrospective cohort of 30 grade II IDHmut+/1p19qcodel oligodendrogliomas. The prognostic value of MCM6 mRNA level was confirmed in The Cancer Genome Atlas (TCGA) IDHmut+/1p19qcodel gliomas. The transcriptomic approach revealed that high transcriptional expressions of MCM6 and MKI67 were both linked positively with cell cycle progression, DNA replication, mitosis, pro-neural phenotype as well as neurogenesis, and negatively with microglial cell activation, immune response, positive regulation of myelination, oligodendrocyte development, beta-amyloid binding, and postsynaptic specialization. In conclusion, the overexpression of MCM6 and/or Ki-67 is independently associated to shorter overall survival in IDHmut+/1p19qcodel AO. These two easy-to-use and cost-effective markers could thus be used concurrently in routine pathology practice. Additionally, the transcriptomic analyses showed that AO with high proliferation index have down-regulated immune response and lower microglial cells activation, and bears pro-neural phenotype.

Breast cancer is the most common malignancy diagnosed during pregnancy. Strong data on the genomic profile of pregnancy-associated breast cancer are lacking. This systematic review aims to integrate and analyze all existing data from the literature regarding the genomic background and the gene mutational patterns of pregnancy-associated breast cancer. Using various genomic analysis methods, multiple differentially expressed genes and numerous non-silent mutations have been detected. More particularly, our review demonstrates the aberrant expression of several oncogenes (e.g., MYC, SRC, FOS), tumor suppressor genes (e.g., TP53, PTEN, CAV1), apoptosis regulators (e.g., PDCD4, BCL2, BIRC5), transcription regulators (e.g., JUN, KLF1, SP110), genes involved in DNA repair mechanisms (e.g., Sig20, BRCA1, BRCA2, FEN1), in cell proliferation (e.g., AURKA, MKI67), in the immune response (e.g., PD1, PDL1), and in other significant biological processes (e.g., protein modification, internal cell motility). Further research on the genomic profile of pregnancy-associated breast cancer is urgently required in order to identify potential biomarkers facilitating early-stage diagnosis and individualized therapy.

Keywords: breast; cancer; gene; mutation; pregnancy.

In female mammals, the abnormal apoptosis of ovarian granulosa cells (GCs) impairs follicular development and causes reproductive dysfunction. Many studies have indicated that the FGFR1 gene of the PI3K signaling pathway and the p65 subunit of the transcription factor NF-κB may regulate the proliferation and apoptosis of GCs involved in follicular development. However, little is known about whether p65 regulates the transcription of FGFR1, as well as the biological effects of p65 and FGFR1 on the survival of GCs and follicular development. In porcine follicles and GCs, we found that p65 and FGFR1 were exclusively expressed in the GCs of follicles, and the mRNA and protein levels of p65 and FGFR1 significantly increased from small to large follicles. Both p65 and FGFR1 were found to activate the PI3K signaling pathway, and the expressions of proliferation markers (PCNA and MKI67) and the anti-apoptotic gene BCL2 were significantly increased by p65 and FGFR1. Furthermore, both p65 and FGFR1 were observed to promote cell proliferation and inhibit the cell apoptosis of GCs, and p65 was confirmed to bind at the -348/-338 region of FGFR1 to positively regulate its transcription. Moreover, p65 was further found to enhance the pro-proliferation and anti-apoptotic effects of FGFR1. Taken together, p65 may target the -348/-338 region of FGFR1, promote the transcription of FGFR1, and enhance the pro-proliferation effect and anti-apoptotic effect of FGFR1 to facilitate the growth of follicles. This study will provide useful information for further investigations on the p65-mediated-FGFR1 signaling pathway during folliculogenesis in mammals.

Previous reports have suggested a link between pulmonary tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), and the development of lung adenocarcinoma (LUAD) and sarcoidosis. Furthermore, these lung diseases share certain clinical similarities that can challenge differential diagnosis in some cases. Here, through comparison of lung transcriptome-derived molecular signatures of TB, LUAD and sarcoidosis patients, we identify certain shared disease-related expression patterns. We also demonstrate that MKI67, an over-expressed gene shared by TB and LUAD, is a key mediator in Mtb-promoted tumor cell proliferation, migration, and invasion. Moreover, we reveal a distinct ossification-related TB lung signature, which may be associated with the activation of the BMP/SMAD/RUNX2 pathway in Mtb-infected macrophages that can restrain mycobacterial survival and promote osteogenic differentiation of mesenchymal stem cells. Taken together, these findings provide novel pathogenic links and potential molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.

Gastric cancer (GC) is one of the most fatal common cancers in worldwide. Helicobacter pylori (H. pylori) infection is closely related to the development of GC, although the mechanism is still unclear. In our study, we aim to develop a robust messenger RNA (mRNA) signature associated with H. pylori (-) GC that can sensitively and efficiently predict the prognostic. The RNA-seq expression profile and corresponding clinical data of 598 gastric cancer samples and 63 normal samples obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database. Using gene set enrichment analysis H. pylori (+) GC and H. pylori (-) GC patients and normal samples to select certain genes for further analysis. Using univariate and multivariate Cox regression model to establish a gene signature for predicting the overall survival (OS). Finally, we identified G2/M related seven-mRNA signature (TGFB1, EGF, MKI67, ILF3, INCENP, TNPO2, and CHAF1A) closely related to the prognosis of patients with H. pylori (-) GC. The seven-mRNA signature was identified to act as an independent prognostic biomarker by stratified analysis and multivariate Cox regression analysis. It was also validated on two test groups from TCGA and GSE15460 and shown that patients with high-risk scores based on the expression of the seven mRNAs had significantly shorter survival times compared to patients with low-risk scores (P < .0001). In this study, we developed a seven-mRNA signature related to G2/M checkpoint from H. pylori (-) GCs that as an independent biomarker potentially with a good performance in predicting OS and might be valuable for the clinical management for patients with GC.