The Consequence of Oncomorphic TP53 Mutations in Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy, with an alarmingly poor prognosis attributed to late detection and chemoresistance. Initially, most tumors respond to chemotherapy but eventually relapse due to the development of drug resistance. Currently, there are no biological markers that can be used to predict patient response to chemotherapy. However, it is clear that mutations in the tumor suppressor gene TP53, which occur in 96% of serous ovarian tumors, alter the core molecular pathways involved in drug response. One subtype of TP53 mutations, widely termed gain-of-function (GOF) mutations, surprisingly converts this protein from a tumor suppressor to an oncogene. We term the resulting change an oncomorphism. In this review, we discuss particular TP53 mutations, including known oncomorphic properties of the resulting mutant p53 proteins. For example, several different oncomorphic mutations have been reported, but each mutation acts in a distinct manner and has a different effect on tumor progression and chemoresistance. An understanding of the pathological pathways altered by each mutation is necessary in order to design appropriate drug interventions for patients suffering from this deadly disease.     

Sanger Sequencing for BRCA1 c.68_69del,BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.     

Sirt3, Mitochondrial ROS, Ageing, and Carcinogenesis

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.     

KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.     

KRAS/BRAF Analysis in Ovarian Low-Grade Serous Carcinoma Having Synchronous All Pathological Precursor Regions

Ovarian low-grade serous carcinoma is thought to begin as a serous cystadenoma or adenofibroma that progresses in a slow stepwise fashion. Among the low-grade serous carcinomas, there is a high frequency of activating mutations in the KRAS or BRAF genes; however, it remains unclear as to how these mutations contribute to tumor progression. This is the first report to track the histopathological progression of serous adenofibroma to low-grade serous carcinoma. Each stage was individually analyzed by pathological and molecular genetic methods to determine what differences occur between the distinct stages of progression.     

Triterpenoid Saponins from Stauntonia chinensis Ameliorate Insulin Resistance via the AMP-Activated Protein Kinase and IR/IRS-1/PI3K/Akt Pathways in Insulin-Resistant HepG2 Cells

Inflammation and oxidative stress play crucial roles in the etiology of type 2 diabetes mellitus. In this study, we examined the anti-diabetic effects of triterpenoid saponins extracted from Stauntonia chinensis on stimulating glucose uptake by insulin-resistant human HepG2 cells. The results showed that saponin 6 significantly increased glucose uptake and glucose catabolism. Saponin 6 also enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and activated the insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway. Therefore, our results suggest that saponins from S. chinensis improve glucose uptake and catabolism in hepatic cells by stimulating the AMPK and the IR/IRS-1/PI3K/Akt signaling pathways. The results also imply that saponins from S. chinensis can enhance glucose uptake and insulin sensitivity, representing a promising treatment for type 2 diabetes mellitus.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.     

The Involvement of RhoA and Wnt-5a in the Tumorigenesis and Progression of Ovarian Epithelial Carcinoma

Background

Ras homolog gene family member A (RhoA) is involved in Wnt-5a–induced migration of gastric and breast cancer cells. We investigated the roles of RhoA and Wnt-5a in ovarian carcinoma.

Methods

RhoA and Wnt-5a mRNA and protein expression in normal fallopian tube epithelium, benign tumors, primary ovarian carcinomas, and metastatic omentum were quantified. RhoA or Wnt-5a was knocked down in OVCAR3 ovarian carcinoma cells using siRNAs and cell phenotype and expression of relevant molecules were assayed.

Results

RhoA and Wnt-5a mRNA and protein expression were found to be significantly higher in metastatic omentum than in ovarian carcinomas, benign tumors, and normal fallopian tube epithelium (p < 0.05), and positively associated with differentiation and FIGO staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). RhoA and Wnt-5a expression were positively correlated in ovarian carcinoma (p = 0.001, R² = 0.1669). RhoA or Wnt-5a knockdown downregulated RhoA and Wnt-5a expression; reduced cell proliferation; promoted G₁ arrest and apoptosis; suppressed lamellipodia formation, cell migration, and invasion; and reduced PI3K, Akt, p70S6k, Bcl-xL, survivin, and VEGF mRNA or protein expression.

Conclusions

This is the first demonstration that RhoA and Wnt-5a are associated with ovarian carcinogenesis and apoptosis inhibition; there might be positive correlation between RhoA and Wnt-5a expression. RhoA is a potential tumorigenesis, differentiation, and progression biomarker in ovarian carcinoma.     

Activation of VCAM-1 and Its Associated Molecule CD44 Leads to Increased Malignant Potential of Breast Cancer Cells

VCAM-1 (CD106), a transmembrane glycoprotein, was first reported to play an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (α4β1). In the present study, we observed that VCAM-1 expression can be induced in many breast cancer epithelial cells by cytokine stimulation in vitro and its up-regulation directly correlated with advanced clinical breast cancer stage. We found that VCAM-1 over-expression in the NMuMG breast epithelial cells controls the epithelial and mesenchymal transition (EMT) program to increase cell motility rates and promote chemoresistance to doxorubicin and cisplatin in vitro. Conversely, in the established MDAMB231 metastatic breast cancer cell line, we confirmed that knockdown of endogenous VCAM-1 expression reduced cell proliferation and inhibited TGFβ1 or IL-6 mediated cell migration, and increased chemosensitivity. Furthermore, we demonstrated that knockdown of endogenous VCAM-1 expression in MDAMB231 cells reduced tumor formation in a SCID xenograft mouse model. Signaling studies showed that VCAM-1 physically associates with CD44 and enhances CD44 and ABCG2 expression. Our findings uncover the possible mechanism of VCAM-1 activation facilitating breast cancer progression, and suggest that targeting VCAM-1 is an attractive strategy for therapeutic intervention.     

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.     

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer.     

Ectopic Expression of CsCTR1, a Cucumber CTR-Like Gene,Attenuates Constitutive Ethylene Signaling in an Arabidopsis ctr1-1 Mutant and Expression Pattern Analysis of CsCTR1 in Cucumber (Cucumis sativus)

The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.     

Risk-Association of Five SNPs in TOX3/LOC643714 with Breast Cancer in Southern China

The specific mechanism by which low-risk genetic variants confer breast cancer risk is currently unclear, with contradictory evidence on the role of single nucleotide polymorphisms (SNPs) in TOX3/LOC643714 as a breast cancer susceptibility locus. Investigations of this locus using a Chinese population may indicate whether the findings initially identified in a European population are generalizable to other populations, and may provide new insight into the role of genetic variants in the etiology of breast cancer. In this case-control study, 623 Chinese female breast cancer patients and 620 cancer-free controls were recruited to investigate the role of five SNPs in TOX3/LOC643714 (rs8051542, rs12443621, rs3803662, rs4784227, and rs3112612); Linkage disequilibrium (LD) pattern analysis was performed. Additionally, we evaluated how these common SNPs influence the risk of specific types of breast cancer, as defined by estrogen receptor (ER) status, progesterone receptor (PR) status and human epidermal growth factor receptor 2 (HER2) status. Significant associations with breast cancer risk were observed for rs4784227 and rs8051542 with odds ratios (OR) of 1.31 ((95% confidence intervals (CI), 1.10–1.57)) and 1.26 (95% CI, 1.02–1.56), respectively, per T allele. The T-rs8051542 allele was significantly associated with ER-positive and HER2-negative carriers. No significant association existed between rs12443621, rs3803662, and rs3112612 polymorphisms and risk of breast cancer. Our results support the hypothesis that the applicability of a common susceptibility locus must be confirmed among genetically different populations, which may together explain an appreciable fraction of the genetic etiology of breast cancer.