CCN2 induces cellular senescence in fibroblasts

The expression of Ccn2 (CTGF) has been linked to fibrosis in many tissues and pathologies, although its activities in fibroblastic cells and precise mechanism of action in fibrogenesis are still controversial. Here, we showed that CCN2 can induce cellular senescence in fibroblasts both in vitro and in vivo, whereupon senescent cells express an anti-fibrotic “senescence-associated secretory phenotype” (SASP) that includes upregulation of matrix metalloproteinases and downregulation of collagen. Mechanistically, CCN2 induces fibroblast senescence through integrin α₆β₁-mediated accumulation of reactive oxygen species, leading to activation of p53 and induction of p16^INK4a. In cutaneous wound healing, Ccn2 expression is highly elevated only during the initial inflammatory phase and quickly declines thereafter to a low level during the proliferation and maturation phases of healing when myofibroblasts play a major role. Consistent with this expression kinetics, knockdown of Ccn2 has little effect on the rate of wound closure, formation of senescent cells, or collagen content of the wounds. However, application of purified CCN2 protein on cutaneous wounds leads to induction of senescent cells, expression of SASP, and reduction of collagen content. These results show that CCN2 can induce cellular senescence in fibroblasts and is capable of exerting an anti-fibrotic effect in a context-dependent manner.     

Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

MicroRNA Biogenesis Pathway Gene Polymorphisms Are Associated with Breast Cancer Risk

MicroRNAs (miRNAs) play an important role as epigenetic regulators in cancer initiation and progression. One of the mechanisms of miRNA dysregulation is altered functioning of proteins involved in miRNA processing machinery. It has been suggested that single nucleotide polymorphisms (SNPs) within miRNA gene regions, miRNA target genes, and miRNA machinery genes may affect the miRNAs regulation. We selected 25 SNPs in the key genes of miRNA biosynthesis, including DROSHA/RNASEN, DGCR8, DICER1, XPO5, RAN, PIWIL1/HIWI, AGO1/EIF2C1, AGO2, GEMIN4, GEMIN3/DDX20, and DDX5, and investigated the association between these SNPs and the risk of breast cancer. The total number of breast cancer cases and cancer-free controls enrolled in the investigation were 778 (417 breast cancer patients and 361 healthy women). We found that rs11060845 and rs10773771 in the PIWIL1 gene, rs3809142/RAN, rs10719/DROSHA, rs1640299/DGCR8, rs563002/DDX20, rs595055/AGO1, and rs2740348/GEMIN4 were associated with breast cancer risk in Russians.     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Mammaglobin in peripheral blood and the tumor of breast cancer patients

Currently, no molecular biological markers do exist for early diagnosis of breast cancer. One of the possible candidates for the marker of early breast cancer is mammaglobin (MGB1) or SCGB2A2 (secretoglobin, family 2A, member 2), characterized by the maximal expression level in early breast cancer. Using the RT-PCR method MGB1 mRNA expression was examined in 57 tumor tissue samples and 57 samples of morphologically non-malignant tissue (MNT) of breast cancer (BC) patients. Specificity and sensitivity of the MGB1 mRNA assay in peripheral blood of BC patients was evaluated by nested PCR. 169 blood samples (from 95 BC patients, 22 from patients with benign breast tumors, 28 from patients with tumors of other localizations, and 24 samples from healthy donors) have been analyzed. MGB1 expression was significantly higher in BC tissue samples compared to MNT (p = 0.0019). The maximal expression level was in the samples T1 (p = 0.013), stage I BC (p = 0.037), GI (p = 0.0019). MGB1 expression positively correlated with expression of estrogen (p = 0.034) and progesterone (p = 0.0004) receptors. Sensitivity and specificity of the MGB1 mRNA assay in peripheral blood were 60.6 and 92.3%, respectively. Expression of MGB1 was higher in BC than MNT and it decreased during BC progression. The sensitivity and specificity of the MGB1 mRNA assay may be used as an additional diagnostic method.     

Association of <Emphasis Type="Italic">PALB2</Emphasis> sequence variants with the risk of early-onset breast cancer in patients from Turkey

The PALB2 gene, has been accepted as a moderate-penetrance gene associated with breast cancer susceptibility and this gene product is involved in the DNA damage repair pathway via co-localization with BRCA2. Germline PALB2 mutations are associated with an increased breast cancer risk. However, the prevalence of the diverse types of PALB2 variants depend on the population. Thus, the aim of the present study was to determine, for the first time, the prevalence of PALB2 variants in a Turkish population of BRCA1/BRCA2-negative early-onset patients with breast cancer. In total, 223 Turkish patients with BRCA1/BRCA2 negative early-onset breast cancer and 60 unaffected women were included in the study. All the coding exons and intron/exon boundaries of PALB2 were subjected to mutational analysis by heteroduplex analysis (HDA)and DNA sequencing. Eighteen PALB2 variants were found in breast cancer patients within the Turkish population. Three variants (c.271G>A, c.404C>A and c.2981T>A) have not been previously reported. In addition, nine intronic variants were described, and this study is the first to describe the c.1685-44T>A intronic variant. The prevalence of possible pathogenic PALB2 variants was found to be 4.03 % in BRCA1/2-negative Turkish patients with early-onset breast cancer. Different variants of PALB2 have been reported in the literature, and the prevalence of these variants could different for each population. This is the first study to investigate the prevalence of PALB2 variants in Turkish patients with early-onset breast cancer.     

Associations of MMP-2 −1306 C/T and MMP-9 −1562 C/T polymorphisms with breast cancer risk among different populations: a meta-analysis

The meta-analysis aims to investigate association between two matrix metalloproteinases (MMPs) polymorphisms (MMP-2 ?1306 C/T and MMP-9 ?1562 C/T) and breast cancer risk. Eligible studies were retrieved from relevant databases, based on predefined criteria. Quality assessment was evaluated by Newcastle–Ottawa Scale. Odds ratio (OR) with its 95% confidence interval (CI) was selected as the effect size for the meta-analysis. As a result, 13 studies were included. MMP-2 ?1306 C/T polymorphism was not significantly associated with breast cancer risk under all genetic models (P > 0.05). However, subgroup analysis stratified by ethnicity showed a significant association between MMP-2 ?1306 C/T polymorphism and reduced breast cancer risk in Asian populations under allelic model (OR 0.60, 95% CI 0.39–0.90, P = 0.02) and dominant model (OR 0.55, 95% CI 0.34–0.89, P = 0.02). MMP-9 ?1562 C/T polymorphism was significantly related to increased breast cancer risk under allelic model (OR 1.50, 95% CI 1.06–2.12, P = 0.02), additive model (OR 1.45, 95% CI 1.02–2.05, P = 0.04) and recessive model (OR 1.54, 95% CI 1.13–2.12, OR 0.007). A significant association between MMP-9 ?1562 C/T polymorphism and increased breast cancer risk in Caucasian was detected under most of the genetic models (P < 0.05). MMP-2 ?1306 C/T polymorphism might be significantly associated with reduced breast cancer risk in Asian, while MMP-9 ?1562 C/T might be closely related to increased breast cancer risk, especially in Caucasian.     

Cereal cyst nematode resistance gene <Emphasis Type="Italic">CreV</Emphasis> effective against <Emphasis Type="Italic">Heterodera filipjevi</Emphasis> transferred from chromosome 6VL of <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> to bread wheat

Cereal cyst nematodes (CCN) are a global economic problem for cereal production. Heterodera filipjevi is one of the most commonly identified and widespread CCN species found in many wheat production regions of the world. Transferring novel genes for resistance to H. filipjevi from wild relatives of wheat is a promising strategy for protection of wheat crops. A set of wheat–Dasypyrum villosum chromosome addition lines, T6V#4S·6AL translocation lines and their donor parental lines were tested for their response to the nematode. D. villosum and wheat–D. villosum disomic addition line DA6V#4 were resistant. As T6V#4S·6AL translocation lines were susceptible, resistance was presumed to be located on chromosome 6V#4L. The objective of this study was to produce and characterize wheat–6V#4L translocations and confirm the chromosome location of the resistance. Introgression lines T6V#4L·6AS, T6V#4L-4BL·4BS and DT6V#4L were developed and subjected to molecular cytogenetic analysis. These and four additional wheat–6V#4 introgression lines were tested for response to H. filipjevi in the greenhouse. The results indicated that introgression lines DA6V#4, T6V#4L·6AS, T6V#4L-4BL·4BS, T6V#4L·6V#4S-7BS and DT6VL#4 had higher levels of H. filipjevi resistance than their recurrent parent. However, Del6V#4L-1 and translocation line T6V#4S·6AL were equally susceptible to wheat cv. Chinese Spring. The CCN resistance gene, temporarily named CreV, was therefore physically mapped to chromosome arm 6V#4L FL 0.80–1.00. Translocation chromosomes T6V#4L·6AS transferred to a modern wheat cv. Aikang 58 with its co-dominant molecular markers could be utilized as a novel germplasm for CCN resistance breeding in wheat.     

Signature of positive selection of <Emphasis Type="Italic">PTK6</Emphasis> gene in East Asian populations: a cross talk for <Emphasis Type="Italic">Helicobacter pylori</Emphasis> invasion and gastric cancer endemicity

Analysis of natural selection events is an attractive strategy for identification of functional variants shaped by gene–environmental interactions and human adaptation. Here, we identified PTK6, a Src-related tyrosine kinase gene, underlying positive selection in East Asian populations. Interestingly, PTK6 variant showed significant correlation with gastric cancer incidences which was the highest in East Asian populations. The high prevalence of gastric cancer in East Asians was also believed to be strongly affected by Helicobacter pylori infection and dietary habit. Therefore, we speculated a competitive interaction of cancer-associated molecules for activation/reduction, where PTK6 likely plays a role through CagA-driven signaling pathway after H. pylori infection. This hypothesis was also supported by our gene expression analysis and the dating of the selective event which was estimated to be ~16,500 years ago, much later than H. pylori invasion in human 50,000 years ago. Establishment of cross talk between PTK6 and CagA by functional studies may further elucidate the underlying biology of H. pylori-mediated gastric cancer.     

Downregulation of expression of mater genes <Emphasis Type="Italic">SOX9, FOXA2</Emphasis>, and <Emphasis Type="Italic">GATA4</Emphasis> in pancreatic cancer cells stimulated with TGFβ1 epithelial–mesenchymal transition

We show characteristic morphological changes corresponding to epithelial–mesenchymal transition (EMT) program fulfillment in PANC1 cell line stimulated with TGFβ1. Our results support downregulation of E-cadherin protein. We show 5- and 28-fold increase in SNAI1 and SNAI2 expression levels and 25- and 15-fold decrease in CDH1 and KRT8 expression levels, respectively, which confirms the EMT-program fulfillment. We demonstrate downregulation of expression of pancreatic master genes SOX9, FOXA2, and GATA4 (2-, 5-, and 4-fold, respectively) and absence of significant changes in HES1, NR5A2, and GATA6 expression levels in the cells stimulated with TGFβ1. Our results indicate the absence of induction of expression of PTF1A, PDX1, HNF1b, NEUROG3, RPBJL, NKX6.1, and ONECUT1 genes, which are inactive in PANC1 cell line after the EMT stimulated by TGFβ1.     

Inhibitory Effect of Vaginal Lactobacillus Supernatants on Cervical Cancer Cells

Lactobacilli have been shown to inhibit the proliferation of several types of cancer cells, but the effects of vaginal Lactobacilli on cervical cancer cells have seldom been reported. We incubated Caski cells with supernatants of predominant strains in the vagina and investigated their effects on cell growth and the possible mechanisms. Cell-free supernatants of Lactobacillus crispatus, L. jensenii, and L. gasseri were prepared and purified. Caski cells were treated with various concentrations of Lactobacillus supernatants (LS). The effect of LS on cell growth was investigated using MTT assays. The influence of LS on the cell cycle and expression of human papillomavirus (HPV) E6 and E7 oncogenes was determined by flow cytometry and RT-PCR, respectively. LS-inhibited Caski cell proliferation caused morphological changes in a pH-independent manner. Flow cytometric analysis revealed that cells exposed to LS exhibited a significant increase of cell number in S phase and a strong decrease of cell number in G2/M phase. Expression of HPV E6 and E7 oncogenes, as well as CDK2 and cyclin A was decreased after treatment with LS, while expression of p21 was increased. Supernatants of L. crispatus, L. jensenii, and L. gasseri have inhibitory effects on the viability of cervical cancer cells via regulation of HPV oncogenes and cell cycle-related genes. Lactobacillus, as a promising treatment for cancer, is being assessed for its effect, and these results provide further evidence in this respect.     

Analysis of the conservation of synteny between Fugu and human chromosome 12

Background

The pufferfish Fugu rubripes (Fugu) with its compact genome is increasingly recognized as an important vertebrate model for comparative genomic studies. In particular, large regions of conserved synteny between human and Fugu genomes indicate its utility to identify disease-causing genes. The human chromosome 12p12 is frequently deleted in various hematological malignancies and solid tumors, but the actual tumor suppressor gene remains unidentified.

Results

We investigated approximately 200 kb of the genomic region surrounding the ETV6 locus in Fugu (fETV6) in order to find conserved functional features, such as genes or regulatory regions, that could give insight into the nature of the genes targeted by deletions in human cancer cells. Seven genes were identified near the fETV6 locus. We found that the synteny with human chromosome 12 was conserved, but extensive genomic rearrangements occurred between the Fugu and human ETV6 loci.

Conclusion

This comparative analysis led to the identification of previously uncharacterized genes in the human genome and some potentially important regulatory sequences as well. This is a good indication that the analysis of the compact Fugu genome will be valuable to identify functional features that have been conserved throughout the evolution of vertebrates.

     

Characterization and prognosis of estrogen receptor-positive/progesterone receptor-negative male breast cancer: a population-based study

Background

The aim of this study was to explore the characteristics and prognostic information of estrogen receptor-positive/progesterone receptor-negative (ER+/PR?) male breast cancer.

Methods

Using the US National Cancer Institute’s Surveillance, Epidemiology, and End Results database, we compared the demographics, clinical characteristics, and outcome of estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) patients with ER+/PR? male breast cancer patients from 1990 to 2010. Two thousand three hundred twenty-two patients with ER+/PR+ tumors and 355 patients with ER+/PR? tumors were included in our study.

Results

ER+/PR? patients were younger (P?=?0.008) and more likely to be African American (P?<?0.001) while presented with higher histological grade (P?<?0.001), larger tumor size (P?=?0.010), and more invasion to the lymph nodes (P?=?0.034) and distant sites (P?<?0.001), thus later stage (P?=?0.001). Despite higher chance of receiving chemotherapy (51.0% vs 36.5%, P?<?0.001), ER+/PR? patients experienced significantly worse breast cancer-specific survival (BSCC) (P?<?0.001) and shorter overall survival (OS) (P?=?0.003). Multivariate Cox model confirmed that tumor size, lymph node invasion, metastasis, and surgery were independent prognostic factors of both BSCC and OS for ER+/PR? male breast cancer. Age at diagnosis and chemotherapy were significantly associated with OS but not with BSCC.

Conclusion

ER+/PR? male breast cancer was more aggressive and experienced shorter survival than ER+/PR+ patients. The prognosis was mainly associated with tumor size, lymph node invasion, metastasis, and surgery.