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.     

Overexpression of the recently identified oncogene <Emphasis Type="Italic">REDD1</Emphasis> correlates with tumor progression and is an independent unfavorable prognostic factor for ovarian carcinoma

Background

Regulated in development and DNA damage response (REDD1), a gene responding to hypoxia or multiple DNA damage events, was recently implicated in cancer development and progression. Previously, in vivo and in vitro experiments indicated that REDD1 functions as an oncogene in ovarian cancer cells. However, the role of REDD1 in cancer cell migration and invasion and in clinical significance of prognostic values is not examined in detail.

Methods

We detected the REDD1 protein expression by immunohistochemistry in 18 normal ovarian surface epithelium or fallopian tube epithelium specimens, 24 ovarian borderline tumors, and 229 ovarian cancers. Fisher’s exact test, logistic regression analysis, the Kaplan–Meier method, and the log-rank test were used to evaluate the association of REDD1 with clinical factors, overall survival and disease-free survival. The prognostic predictive value of REDD1 for ovarian cancer patients was evaluated using multivariate Cox proportional hazard regression models. REDD1 expression in HEY, HEY A8, SKOV3, SKOV3 ip1, OVCA429, OVCA433 and A2780 human ovarian epithelial cancer cell lines was detected by western blotting. The role of REDD1 in cell invasion and migration was assessed by transwell migration and invasion assays using SKOV3, A2780, HEY, HEYA8, and SKOV3-REDD1 with parental A2780-REDD1 HEY-REDD1i and HEY A8-REDD1i.

Results

High expression of REDD1 was observed in 35.4% of primary ovarian carcinoma samples. Overexpression of cytoplasmic REDD1 in ovarian cancer was significantly associated with serous carcinoma (P?<?0.001), late-stage disease (P?<?0.001), ascites (P?<?0.001), and partial or non-response to chemotherapy (P?<?0.001). High cytoplasmic expression of REDD1 was correlated with poorer overall survival (P?<?0.001) and disease-free survival (P?<?0.001). The multivariate Cox proportional hazards regression analysis indicated that patients with high cytoplasmic REDD1 expression had a high risk of death (P?<?0.001) and high risk of an event (i.e., recurrence, progression, or death) (P?<?0.001). REDD1 was first reported as an independent prognostic factor in ovarian cancer patients. In addition, REDD1 overexpression enhanced ovarian cancer cell migration and invasion.

Conclusion

REDD1 is an independent unfavorable prognostic factor in ovarian carcinoma and may promote ovarian cancer metastasis.

     

Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression,and activation of their potential target genes in clear cell renal cell carcinoma

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31–48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher’s exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher’s exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman’s correlation coefficient r _s ranging 0.31–0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (r _s ranging 0.35–0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.     

MYO18B promotes hepatocellular carcinoma progression by activating PI3K/AKT/mTOR signaling pathway

Background

MYO18B has been identified as a novel tumor suppressor gene in several cancers. However, its specific roles in the progression of hepatocellular carcinoma (HCC) has not been well defined.

Methods

We firstly identified the expression and prognostic values of MYO18B in HCC using TCGA cohort and our clinical data. Then, MYO18B knockdown by RNA inference was implemented to investigate the effects of MYO18B on HCC cells. Quantitative RT-PCR and Western blot were used to determine gene and protein expression levels. CCK-8 and colony formation assays were performed to examine cell proliferation capacity. Wound healing and transwell assays were used to evaluate the migration and invasion of HepG2 cells.

Results

MYO18B was overexpressed and correlated with poor prognosis in HCC. MYO18B expression was an independent risk factor for overall survival. Knockdown of MYO18B significantly inhibited the proliferation, migration and invasion of HepG2 cells. Meanwhile, MYO18B knockdown could effectively suppress the phosphorylation of PI3K, AKT, mTOR and P70S6K, suggesting that MYO18B might promote HCC progression by targeting PI3K/AKT/mTOR signaling pathway.

Conclusions

MYO18B promoted tumor growth and migration via the activation of PI3K/AKT/mTOR signaling pathway. MYO18B might be a promising target for clinical intervention of HCC.

     

The crucial role of SEMA3F in suppressing the progression of oral squamous cell carcinoma

Background

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancy. Semaphorin 3F (SEMA3F) is highly conserved but present at a lower level in various cancers than in healthy tissues. While it has been reported that SEMA3F is involved in cancer cell proliferation, migration and invasion, its function in OSCC remains unknown.

Methods

The expression of SEMA3F in OSCC tissues and OSCC-derived cells was analyzed using qRT-PCR and western blotting. Using SAS and HSC2 cells, we also monitored the effect of SEMA3F on OSCC cell proliferation, migration and invasion using MTT, colony formation and transwell assays. The function of SEMA3F in OSCC tumor formation was also assessed in vivo.

Results

SEMA3F was significantly downregulated in OSCC tissues and OSCC-derived cells. SEMA3F shows growth inhibitory activity in SAS and HSC2 cells and may act as a tumor suppressor. It can inhibit the migration and invasion potential of OSCC cells. Our results also demonstrate that SEMA3F can suppress the growth of OSCC cells in vivo.

Conclusions

This study revealed that SEMA3F plays a role as a tumor suppressor in OSCC cell proliferation, migration and invasion. Our finding provides new insight into the progression of OSCC. Therapeutically, SEMA3F has some potential as a target for OSCC treatment, given sufficient future research.

     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> mutations and clinical interpretation in 398 ovarian cancer patients: comparison with breast cancer variants in a similar population

Background

Ovarian cancer is the leading cause of death worldwide among gynecologic malignancies. The recent approval of inhibitors of poly (ADP-ribose) polymerase (iPARP) in the treatment of ovarian cancer in the presence of a BRCA1/2 mutation has sparked the analysis of women with such diagnosis, which can further benefit from the detection of carriers in the family. Germline sequence and large rearrangements for BRCA1/2 were tested in 398 consecutive epithelial ovarian cancer (EOC) patients.The aim of this study was to identify the frequency and spectrum of germline BRCA1/2 pathogenic alterations in a cohort of patients with ovarian serous carcinoma, with a view to adequately selecting patients for prevention through family counseling and correlating this frequency with platinum sensitivity as a guidance to identify patients eligible for iPARP in our population.

Results

A total of 96 patients carried a pathogenic germline mutation, accounting for an overall 24.1% mutation incidence. Among mutation carriers, BRCA1 showed 62.5% incidence, BRCA2 rendered 36.5%, and one patient exhibited a mutation in both genes. Three pathogenic mutations were recurrent mutations detected five, three, and four times and represented 12.5% of the mutated samples. Worth highlighting, a 50% mutation incidence was detected when breast and ovarian cancer coexisted in the same patient. Novel mutations amounted to 9.4% of the total mutations, as compared to 4.7% in breast cancer. Forty out of 60 BRCA1 mutations were beyond the ovarian cancer cluster region (OCCR), in stark contrast with 22 out of 36 BRCA2 mutations being inside the OCCR. Taken together, germline BRCA1/2 mutations in EOC patients showed a distinct mutational spectrum compared to our previously published data on breast cancer patients.

Conclusions

In sum, our study provides novel data on ovarian BRCA1/2 mutation prevalence worldwide, enhances adequate patient selection for family counseling and prevention, and sheds light on the benefits of iPARP treatment.

     

Anti-inflammatory effects of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> extracts on human blood mononuclear cells and murine macrophages

The microalgae Phaeodactylum tricornutum (PT) is known for its high content of omega-3 fatty acids, which are known to attenuate inflammation. Additionally, this microalga contains other nutrients such as carbohydrates, vitamins, proteins, and carotenoids and therefore could be of interest for animal and human nutrition. Here, we investigated the effects of hexane, ethanolic, and aqueous extracts on lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) and on the murine macrophage cell line RAW 264.7. Further, the cytotoxicity of the extracts was studied using the MTT assay. The production of pro-inflammatory cytokines was significantly inhibited by the ethanolic and aqueous P. tricornutum extracts but not by the hexane extract. Both at the mRNA and at the protein levels, the aqueous extract inhibited LPS-induced IL-6, IL-1β, TNFα, and COX-2 expression and release by up to 96% (mRNA) and 79% (protein) in a dose-dependent manner. Compared to the aqueous extract, the ethanolic extract was less effective in cytokine inhibition. The production of nitric oxide in RAW 264.7 cells was significantly reduced by all extracts. We showed that the anti-inflammatory effect of P. tricornutum is exerted through inhibition of nuclear factor-κB activation and dependent on the mitogen-activated protein (MAP) kinase pathway. Our data indicate anti-inflammatory effects of the aqueous P. tricornutum extract and provide a basis information on the safety and potential health benefits of P. tricornutum usage for future animal and human nutrition.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

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.

     

On the multicomponent nature of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> flagella

Filaments of the flagellum of the halophilic archaeon Halobacterium salinarum consist of five flagellins: A1, A2, B1, B2, and B3, which are encoded by five genes localized in tandem in two flgA and flgB operons. While the role of flagellins A1 and A2 has been determined, the role of the proteins, B operon products, is still unclear. A mutant strain of H. salinarum with deleted A and B flagellin genes (ΔflgAΔflgB) has been obtained for the first time. This strain has been used to create and analyze the strains carrying only individual B1 or B3 flagellin genes. Cells of the ΔflgAΔflgB strain were shown to have short filamentous formations, 7–8 nm thick, which we have named as X-filaments. It has been shown that X-filaments consist of a protein immunologically related to flagellins A and B. Expression of the B1 and B3 genes is suppressed in the absence of A1, A2, and B2. It has been shown that flagellins B1 and B3 cannot be substituted for flagellin B2 upon the formation of a curved hook-like structure, which serves as a connecting element between the flagellar filament and the motor axis. The multicomponent nature of flagella is discussed in the light of their possible involvement in other cell processes besides providing motility.     

Molecular and cytogenic analysis of pericentromeric heterochromatin in ovarian nurse cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> subgroup species

Evolutionary rearrangements of pericentromeric heterochromatin among Drosophila melanogaster subgroup species have been investigated. A region-specific DNA library from Drosophila orena ovarian nurse cell chromocenter was obtained by the microdissection of polythene chromosomes. The probe has been localized on chromosomes of ovarian nurse cells of Drosophila melanogaster subgroup species using fluorescent hybridization in situ. Sequences homologous to the sequences of the DNA probe were detected in the chromocenter and pericentromeric regions of D. orena polythene chromosomes, in all pericentromeric regions of other species with several exceptions. There was no labeling on one of the arms of the D. simulans chromosome 2; however, these sequences were present on the telomere of D. erecta chromosome 3 and in regions adjacent to the brightly DAPI-stained heterochromatin blocks of D. yakuba, D. santomea and D. teissieri chromosomes 2 and 3. At the S₆ stage (secondary reticulate nucleus), labeled chromatin can be found mostly within a restricted territory in D. orena nucleus; no such chromatin can be detected throughout the rest of the nucleus. On the contrary, at this stage, in nuclei of other species, labeled DNA is spread diffusely.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Contributions of <Emphasis Type="Italic">TaSUTs</Emphasis> to grain weight in wheat under drought

Key message

The homologous genes to OsSUT1-5 in wheat were identified and detailed analysed. TaSUT1 was the predominant sucrose transporter group and it illustrated the genotypic variations towards drought during grain filling.

Abstract

Sucrose transporters (SUT) play crucial roles in wheat stem water soluble carbohydrate (WSC) remobilization to grain. To determine the major functional SUT gene groups in shoot parts of wheat during grain development, drought tolerant varieties, Westonia and Kauz, were investigated in field drought experiments. Fourteen homologous genes to OsSUT1-5 were identified on five homeologous groups, namely TaSUT1_4A, TaSUT1_4B, TaSUT1_4D; TaSUT2_5A, TaSUT2_5B, TaSUT2_5D; TaSUT3_1A, TaSUT3_1D; TaSUT4_6A, TaSUT4_6B, TaSUT4_6D; TaSUT5_2A, TaSUT5_2B, and TaSUT5_2D, and their gene structures were analysed. Wheat plants above the ground were harvested from pre-anthesis to grain maturity and the stem, leaf sheath, rachis, lemma and developing grain were used for analysing TaSUT gene expression. Grain weight, thousand grain weight, kernel number per spike, biomass and stem WSC were characterized. The study showed that among the five TaSUT groups, TaSUT1 was the predominant sucrose transporting group in all organs sampled, and the expression was particularly high in the developing grain. In contrast to TaSUT1, the gene expression levels of TaSUT2, TaSUT3 and TaSUT4 were lower, except for TaSUT3 which showed preferential expression in the lemma before anthesis. The TaSUT5 gene group was very weakly expressed in all tissues. The upregulated gene expression of TaSUT1 Westonia type in stem and grain reveal a crucial role in stem WSC remobilization to grain under drought. The high TaSUT1 gene expression and the significant correlations with thousand grain weight (TGW) and kernel number per spike demonstrated the contribution in Kauz’s high grain yield in an irrigated environment and high TGW in Westonia under drought stress. Further molecular level identification is required for gene marker development.

     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.