Expression level and methylation status of three tumor suppressor genes, <Emphasis Type="Italic">DLEC1</Emphasis>, <Emphasis Type="Italic">ITGA9</Emphasis> and <Emphasis Type="Italic">MLH1</Emphasis>, in non-small cell lung cancer

Despite therapeutic advances, lung cancer remains one of the most common causes of cancer-related death in the world. There is a need to develop biomarkers of diagnostic and/or prognostic value and to translate findings in basic science research to clinical application. Tumor suppressor genes (TSGs) represent potential useful markers for disease detection, progression and treatment target. We tried to elucidate the role of three 3p21.3 TSGs: DLEC1, ITGA9 and MLH1, in non-small cell lung cancer (NSCLC). We assessed their expression pattern by qPCR in 59 NSCLC tissues and in the matched macroscopically unchanged lung tissues. Additionally, we analyzed gene promoter methylation status by methylation-specific PCR in NSCLC samples. We did not find significant correlations between gene expression and methylation. In case of DLEC1 and ITGA9, expression levels were decreased in 71–78 % of tumor samples and significantly different between tumor and normal tissues (P = 0.0001). It could point to their diagnostic value. ITGA9 could also be regarded as a diagnostic marker differentiating NSCLC subtypes, as its expression level was significantly lower in squamous cell carcinoma (P = 0.001). The simultaneous down-regulation of DLEC1 and ITGA9 was observed in 52.5 % of NSCLCs. MSPs revealed high frequencies of gene promoter methylation in NSCLCs: 84 % for DLEC1 and MLH1 and 57 % for ITGA9. Methylation indexes reflected moderate gene methylation levels: 34 % for ITGA9, 27 % for MLH1 and 26 % for DLEC1. However, frequent simultaneous methylation of the studied genes in more than 50 % of NSCLCs suggests the possibility of consider them as a panel of epigenetic markers.     

<Emphasis Type="Italic">BRAF</Emphasis>/K-<Emphasis Type="Italic">ras</Emphasis> mutation,microsatellite instability,and promoter hypermethylation of <Emphasis Type="Italic">hMLH1</Emphasis>/<Emphasis Type="Italic">MGMT</Emphasis> in human gastric carcinomas

Background The BRAF and K-ras genes are the most frequently mutated oncogenes in various human malignancies. We examined BRAF and K-ras mutations in human gastric cancer, and investigated their relationship with microsatellite instability (MSI) and the hypermethylation of promoter regions in hMLH1 and O ⁶ -methylguanine DNA methyltransferase (MGMT).Methods Sixteen gastric cancer cell lines and 62 gastric cancer tissue samples were screened for BRAF and K-ras mutations by direct sequencing. We also performed a microsatellite assay and investigated methylation status in the promoter regions of hMLH1 and MGMT.Results mutation was not found in any of the cancer cell lines examined. One (1.6%) cancer tissue sample showed a point mutation in the BRAF gene (GT_G GA_G; V599E). K-ras mutation (GG_T GA_T, G12D) was detected in five (31%) gastric cancer cell lines and in 1 (1.6%) gastric cancer tissue sample. In the gastric cancer tissue samples examined, MSI was detected in 23 (37%) samples. Hypermethylated promoter regions in hMLH1 and MGMT, respectively, were detected in 6 (10%) and 13 (21%) gastric cancer tissue samples. Microsatellite stable (MSS) tumors showed frequent lymphatic invasion (P = 0.050).Conclusion Although BRAF mutation has been reported in a variety of other human cancers, it is a rare event in the carcinogenesis and progression/development of gastric cancer.     

<Emphasis Type="Italic">SERPINB5</Emphasis> and <Emphasis Type="Italic">AKAP12</Emphasis>-- Expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma

Background  

Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool.     

Prognostic significance of co-overexpression of the <Emphasis Type="Italic">EGFR</Emphasis>/<Emphasis Type="Italic">IGFBP-2</Emphasis>/<Emphasis Type="Italic">HIF-2A</Emphasis> genes in astrocytomas

Overexpression of the EGFR, IGFBP-2 and HIF-2A genes has been observed in high-grade astrocytomas and these genes seem to be functionally related to one another. This study aimed to define the profile of their expressions, interactions and correlation with clinical features and prognostic significance in microdissected tumor samples from 84 patients with astrocytomas of different grades and from 6 white matter non-neoplasic brain tissue sample. EGFR, IGFBP-2 and HIF-2A gene expression levels were analyzed by quantitative real-time PCR and differed significantly between grades I–IV astrocytic tumors (P < 0.0001, P < 0.0001 and P: 0.0013, respectively) when analyzed by the Kruskal–Wallis test. Grade I astrocytomas presented gene expression levels similar to those encountered in samples of microdissected white matter of non-neoplastic brain tissue Overexpression of the EGFR, IGFBP-2 and HIF-2A genes was significantly associated with lower 2-year survival (P: 0.009, P: 0.0002 and P: 0.008, respectively). Co-overexpression of these genes was strongly associated with high-grade gliomas and lower survival in univariate (P < 0.0001) and multivariate (P: 0.009) analysis, suggesting that the co-expression of the EGFR/IGFBP-2/HIF-2A pathway genes may have a more important clinical and biological impact than the expression of each individual gene alone. These data support the existence of a common pathway involving these genes that could contribute to the design of new target treatments.     

<Emphasis Type="Italic">PALB2</Emphasis> analysis in <Emphasis Type="Italic">BRCA2</Emphasis>-like families

BRCA2 and PALB2 function together in the Fanconi anemia (FA)–Breast Cancer (BRCA) pathway. Mono-allelic and bi-allelic BRCA2 and PALB2 mutation carriers share many clinical characteristics. Mono-allelic germline mutations of BRCA2 and PALB2 are risk alleles of female breast cancer and have also been reported in familial pancreatic cancer, and bi-allelic mutations cause a severe form of Fanconi anemia. In view of these similarities, we investigated whether the prevalence of PALB2 mutations was increased in breast cancer families with the occurrence of BRCA2 associated tumours other than female breast cancer. PALB2 mutation analysis was performed in 110 non-BRCA1/2 cancer patients: (a) 53 ovarian cancer patients from female breast-and/or ovarian cancer families; (b) 45 breast cancer patients with a first or second degree relative with pancreatic cancer; and (c) 12 male breast cancer patients from female breast cancer families. One truncating PALB2 mutation, c.509_510delGA, resulting in p.Arg170X, was found in a male breast cancer patient. We conclude that germline mutations of PALB2 do not significantly contribute to cancer risk in non-BRCA1/2 cancer families with at least one patient with ovarian cancer, male breast cancer, and/or pancreatic cancer.     

<Emphasis Type="Italic">ABI3</Emphasis> ectopic expression reduces <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>cell growth properties while inducing senescence

Background  

Mounting evidence has indicated that ABI3 (ABI family member 3) function as a tumor suppressor gene, although the molecular mechanism by which ABI3 acts remains largely unknown.     

The relationship between <Emphasis Type="Italic">EGFR</Emphasis> gain and <Emphasis Type="Italic">VHL</Emphasis> loss in lung adenocarcinoma and poor patient survival

Background  

The prognosis of lung cancer remains poor and clinically applicable prognostic markers have not yet been satisfactory identified. Several chromosomal copy number alterations (CNAs) have been associated with metastasis, relapse, and survival of patients with lung cancer; however, no study has focused exclusively on identifying CNAs at a gene level. The aim of this study was to identify genes whose CNAs are associated with survival of patients with lung adenocarcinoma.     

<Emphasis Type="Italic">PIK3CA</Emphasis> mutations and <Emphasis Type="Italic">TP53</Emphasis> alterations cooperate to increase cancerous phenotypes and tumor heterogeneity

Background/purpose

The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies.

Experimental design/Methods

We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies.

Results

Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic.

Conclusions

This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

     

Induction of apoptosis by <Emphasis Type="Italic">p53</Emphasis>, <Emphasis Type="Italic">bax</Emphasis>, <Emphasis Type="Italic">bcl-2</Emphasis>, and <Emphasis Type="Italic">p21</Emphasis> expressed in colorectal cancer

Background We investigated the influence of genes on the apoptosis of colorectal tumor cells, based on DNA and mRNA kinetics.Methods In 30 colorectal cancer patients, we examined the mRNA expression of p53, bax, bcl-2, and p21 ^WAF1 , and we also investigated the development of tumor cell apoptosis, using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method.Results TUNEL-positive cells showed a positive correlation with bax (P = 0.010) and a negative correlation with p21 (P = 0.04). We also investigated the relationship between p53 point mutation, p21 immunostaining degree, and apoptosis, based on DNA ladder expression. A remarkable correlation (P = 0.0090) was found between p21 and apoptosis.Conclusions The present study findings suggest that tumor cell apoptosis is (1) strongly inhibited by p21, (2) induced by bax, and (3) influenced by bcl-2, which, presumably, inhibits tumor cell apoptosis.     

Prostate screening uptake in Australian <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> carriers

Men who carry mutations in BRCA1 or BRCA2 are at increased risk for prostate cancer. However the efficacy of prostate screening in this setting is uncertain and limited data exists on the uptake of prostate screening by mutation carriers. This study prospectively evaluated uptake of prostate cancer screening in a multi-institutional cohort of mutation carriers. Subjects were unaffected male BRCA1 and BRCA2 mutation carriers, aged 40–69 years, enrolled in the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab) and who had completed a mailed, self-report follow-up questionnaire 3 yearly after study entry. Of the 75 male carriers in this study, only 26 (35%) had elected to receive their mutation result. Overall, 51 (68%) did not recall having received a recommendation to have prostate screening because of their family history, but 41 (55%) had undergone a prostate specific antigen (PSA) test and 32 (43%) a digital rectal examination (DRE) in the previous 3 years. Those who were aware of their mutation result were more likely to have received a recommendation for prostate screening (43 vs. 6%, p = 0.0001), and to have had a PSA test (77 vs. 43%, p = 0.005) and a DRE (69 vs. 29%, p = 0.001) in the previous 3 years. The majority of unaffected males enrolled in kConFab with a BRCA1/2 mutation have not sought out their mutation result. However, of those aware of their positive mutation status, most have undergone at least one round of prostate screening in the previous 3 years.     

Scorpion venom induces glioma cell apoptosis <Emphasis Type="Italic">in vivo</Emphasis> and inhibits glioma tumor growth <Emphasis Type="Italic">in vitro</Emphasis>

Summary Malignant gliomas are the main brain tumors notoriously resistant to currently available therapies, since they fail to undergo apoptosis upon anticancer treatment. Recent progress on enhanced studies of ion channels involved in glioma cells shed new light on the investigation of glioma cell growth and proliferation. Here we report BmK scorpion venom, a rich resource of various ion channels blockers/modulators, induces cell death of cultured malignant glioma U251-MG cells in vitro specifically at a dose of 10 mg/ml while shows no effect on human hepatocellular carcinoma cells and Chinese hamster ovary cells. The glioma cell death was then determined as apoptosis using 4,6-diamidino-2-phenylindole staining and fluorescence-activated cell sorting analysis. After incubation with BmK venom for 32 and 40 h, 36.20% and 63.08% of U251-MG cells showed apoptosis. Furthermore, BmK venom could significantly inhibit the tumor growth in vitro, which was assessed using U251-MG tumor xenografts on severe combined immunodeficiency mice. The tumor volume of the BmK venom treated mice is nearly 1/8 of that of control after 21 days, and the tumor weight is less than half of that of control. That BmK venom induces apoptosis and inhibits growth of glioma may result from the inhibition and/or modulation of various ion channels in glioma cells.     

Molecular Alterations and Expression Dynamics of <Emphasis Type="Italic">LATS1</Emphasis> and <Emphasis Type="Italic">LATS2</Emphasis> Genes in Non-Small-Cell Lung Carcinoma

Large tumor suppressor (LATS) is an important member of the Hippo pathway which can regulate organ size and cell proliferation. However, very little is known about the expression and clinical significance of LATS in lung cancer especially from this part of the world. We elucidated the frequency of LATS1 &LATS2 promoter hypermethylation (by methylation-specific PCR) and expression (by real-time PCR) in sixty nine (n = 69) Non-Small Cell Lung Cancer (NSCLC) patients and their corresponding normal lung tissue samples. We found promoter hypermethylation frequencies of LATS1 & LATS1to be 66.66% (46/69) and 71% (49/69) in NSCLC tissues. Decreased LATS1 & LATS2 mRNA expression was found in 55% and 66.66% of NSCLC patients. The LATS1 mRNA expression was significantly higher in normal lung tissues. Also, the mRNA levels of LATS1 and LATS2 NSCLC tissues with hypermethylation were significantly lower. Multivariable analysis confirmed that LATS1 under expression increased the hazard of death after adjusting for other clinicopathological factors. Importantly, the loss of LATS1 mRNA expression was associated with overall short survival. LATS1 is an independent prognostic factor and may play an important role in NSCLC progression and may serve as a novel therapeutic target of NSCLC.     

Polymorphisms of the <Emphasis Type="Italic">BRCA2</Emphasis> and <Emphasis Type="Italic">RAD51</Emphasis> Genes in Breast Cancer

Summary We performed a case-control study (150 cases and 150 controls) to test the association between three polymorphisms in BRCA2 and RAD51 genes and breast cancer risk. Genotypes were determined in DNA from blood cells by PCR–RFLP. Cancer occurrence was strongly associated with the BRCA2 Met/1915Thr homozygous polymorphic variants, whereas heterozygous variant was associated with significant reduction in breast cancer risk. Gene-gene interaction between the BRCA2-Met1915Thr Thr/Thr and BRCA2-Met784Val Met/Met homozygous variants increased the risk. Therefore, the Met1915Thr polymorphism in the BRCA2 gene may be considered as an independent marker of breast cancer.     

Managing Patient with Mutations in <Emphasis Type="Italic">PALB2</Emphasis>, <Emphasis Type="Italic">CHEK2</Emphasis>, or <Emphasis Type="Italic">ATM</Emphasis>

Purpose of Review

The use of panel testing of multiple cancer-causing genes has allowed to find a subset of patients with harmful mutations in moderate penetrance genes. While extensive information is available regarding patients with BRCA1 and BRCA2 pathogenic variants, information regarding these less common genes and their management remains scarce. The aim of this review is to discuss penetrance, incidence, and management recommendations for PALB2, ATM, and CHEK2.

Recent Findings

NCCN guidelines now provide management recommendation for patients with pathogenic variants in these genes. In addition, more widespread testing has provided more information on penetrance and incidence. Although this is a huge step toward improving quality of care, prospective studies are still needed. We summarize the NCCN and other guidelines/suggestions for these genes and deliver our insight on the matter based on the best information we could find.

Summary

PALB2, ATM, and CHEK2 are less penetrant than BRCA1–2 and have a different spectrum, suggesting differing management. Data about incidence and penetrance along with management recommendations for these genes are provided.

     

Dysregulated <Emphasis Type="Italic">miR-183</Emphasis> inhibits migration in breast cancer cells

Background  

The involvement of miRNAs in the regulation of fundamental cellular functions has placed them at the fore of ongoing investigations into the processes underlying carcinogenesis. MiRNA expression patterns have been shown to be dysregulated in numerous human malignancies, including breast cancer, suggesting their probable involvement as novel classes of oncogenes or tumour suppressor genes. The identification of differentially expressed miRNAs and elucidation of their functional roles may provide insight into the complex and diverse molecular mechanisms of tumorigenesis. MiR-183 is located on chromosome 7q32 and is part of a miRNA family which are dysregulated in numerous cancers. The aims of this study were to further examine the expression and functional role of miR-183 in breast cancer.     

Large genomic rearrangements of the <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> genes: review of the literature and report of a novel <Emphasis Type="Italic">BRCA1</Emphasis> mutation

Germline mutations in BRCA1 and BRCA2 increase the risk for developing breast and ovarian cancer. Previously, the techniques available allowed only for the identification of small genomic alterations, but the dawn of new technology now allows for the rapid detection of large genomic rearrangements (LGRs). LGRs in BRCA1 are responsible for between 0 and 27% of all BRCA1 disease-causing mutations identified in numerous populations. Such alterations are far less common in the BRCA2 gene. To determine the impact of BRCA1 and BRCA2 LGRs in South Africa, 52 hereditary breast and/or ovarian South African families (36 were Afrikaners) were screened for BRCA1 and BRCA2 LGRs using multiplex ligation-dependent probe amplification. These patients were previously shown to be BRCA1 and BRCA2 small mutation negative. One LGR was detected in BRCA1 in a South African family with Greek ancestry. This is a novel deletion of both exons 23 and 24 (NG_005905.2:g.169527_180579del). This first study of BRCA rearrangements in South Africa reveals that LGRs comprise ~3% of identified BRCA1 mutations, a low rate in comparison to other populations. In addition, we have reviewed all 98 previously characterized BRCA1/2 LGRs and re-named them according to the recommended HGVS nomenclature, using the recently released RefSeqGene records, NG_005905.2 and NG_012772.1 for BRCA1 and BRCA2. A standardized resource is now provided which will assist researchers in determining whether their LGRs are novel. Furthermore, we have clarified some of the previously misunderstood rules of nomenclature, which will make uniform reporting of BRCA1/2 easier in the future.     

Mutation analysis of <Emphasis Type="Italic">PALB2</Emphasis> in <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis>-negative breast and/or ovarian cancer families from Eastern Ontario,Canada

Background

PALB2 has emerged as a breast cancer susceptibility gene. Mutations in PALB2 have been identified in almost all breast cancer populations studied to date, but the rarity of these mutations and lack of information regarding their penetrance makes genetic counseling for these families challenging. We studied BRCA1/2 -negative breast and/or ovarian cancer families to a) assess the contribution of PALB2 mutations in this series and b) identify clinical, pathological and family history characteristics that might make PALB2 screening more efficient.

Methods

The coding region of the PALB2 gene was analyzed in 175 probands with family histories of breast and/or ovarian cancer ascertained from a single Canadian institution in Eastern Ontario.

Results

We identified 2 probands with PALB2 mutations that are known or strongly considered to be pathogenic and 3 probands with missense mutations that are possibly pathogenic. One of the identified truncating mutations [c.3113G > A (p.Gly1000_Trp1038del – major product)], has been previously described while the other four mutations [c.3507_3508delTC (p.H1170Ffs*19), c.1846G > C (p.D616H), c.3418 T > G (p.W1140G), c.3287A > G (p.N1096S)] have not been previously reported. Loss of heterozygosity was detected in two breast tumors from one c.3507_3508delTC mutation carrier but not in other available tumors from that family or in tumors from carriers of other mutations.

Conclusions

PALB2 mutation screening identifies a small, but significant number of mutations in BRCA1/2 -negative breast and/or ovarian cancer families. We show that mutations are more likely to be found in families with three or more breast cancers as well as other BRCA2-related cancers. In our cohort, both clearly pathogenic mutations were identified in premenopausal breast cancer cases (2/77, 2.6%). Testing should be preferentially offered to affected women from such families.