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.     

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.     

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.     

Twist-1 Up-Regulation in Carcinoma Correlates to Poor Survival

Epithelial-to-mesenchymal transition (EMT) facilitates tumor metastasis. Twist is a basic helix-loop-helix protein that modulates many target genes through E-box-responsive elements. There are two twist-like proteins, Twist-1 and Twist-2, sharing high structural homology in mammals. Twist-1 was found to be a key factor in the promotion of metastasis of cancer cells, and is known to induce EMT. Twist-1 participation in carcinoma progression and metastasis has been reported in a variety of tumors. However, controversy exists concerning the correlation between Twist-1 and prognostic value with respect to carcinoma. A systematic review and meta-analysis were performed to determine whether the expression of Twist-1 was associated with the prognosis of carcinoma patients. This analysis included 17 studies: four studies evaluated lung cancer, three evaluated head and neck cancer, two evaluated breast cancer, two evaluated esophageal cancer, two evaluated liver cancer and one each evaluated osteosarcoma, bladder, cervical and ovarian cancer. A total of 2006 patients were enrolled in these studies, and the median trial sample size was 118 patients. Twist-1 expression was associated with worse overall survival (OS) at both 3 years (hazard ratio “HR” for death = 2.13, 95% CI = 1.86 to 2.45, p < 0.001) and 5 years (HR for death = 2.01, 95% CI = 1.76 to 2.29, p < 0.001). Expression of Twist-1 is associated with worse survival in carcinoma.     

Functional Analysis of Familial Asp67Glu and Thr1051Ser BRCA1 Mutations in Breast/Ovarian Carcinogenesis

Estrogen is believed to be pre-initiator in the risk of breast cancer. The BRCA1 is a tumor suppressor gene associated with breast and ovarian cancer risk. This report describes functional analysis of two BRCA1 missense mutations (Asp67Glu and Thr1051Ser) observed in the familial breast/ovarian cancer patients in Thailand. Levels of luciferase activity of the two mutations were relatively lower than in the wild-type BRCA1. It is indicated that mutants may fail to promote the estrogen receptor dependent functions. It is presumed that estrogen and insulin/IGF-1 regulate c-Myc and cyclin D1 during breast cancer cell proliferation. It is also likely to affect ubiquitination mechanism. Since three affected cancer families carry the Asp67Glu mutation, it is believed that this type of mutation could have some effect on breast/ovarian cancer progression.     

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.     

Modulation of P1 and EGF Expression by Baicalin

Mycoplasma pneumoniae (M. pneumoniae) is increasingly recognized as a major cause of acute respiratory tract infections. Today, macrolides are used in the primary treatment of M. pneumoniae infection. However, with the increasing prevalence of strains resistant to macrolides, as well as reports of toxicity and adverse side effects, it is necessary to develop an alternative therapeutic agent. A compound recipe—Qinbaiqingfei pellets (Qinbai)—have already been approved in China as the first effective traditional Chinese medicine to be used against M. pneumoniae. Herein, we characterize the mechanism by which Qinbai interacts with M. pneumoniae and lung epithelial cells. The fact that Baicalin is the key component of Qingbai leads us to believe its study is important to elucidating the mechanism of the action of Qinbai. In this study, we describe the complex impact of Baicalin on the adhesin protein P1 of M. pneumoniae and on the expression of epidermal growth factor (EGF) in BALB/c mice and A549 cells infected with M. pneumonia. We draw the conclusion that Baicalin not only cured M. pneumoniae infection by inhibiting P1 expression, but also enhanced the repair of lung epithelial cells by upregulating EGF. Finally, we demonstrate that Baicalin plays a role in Qinbai treatment.     

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.     

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.     

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.     

NotI Microarrays: Novel Epigenetic Markers for Early Detection and Prognosis of High Grade Serous Ovarian Cancer

Chromosome 3-specific NotI microarray (NMA) containing 180 clones with 188 genes was used in the study to analyze 18 high grade serous ovarian cancer (HGSOC) samples and 7 benign ovarian tumors. We aimed to find novel methylation-dependent biomarkers for early detection and prognosis of HGSOC. Thirty five NotI markers showed frequency of methylation/deletion more or equal to 17%. To check the results of NMA hybridizations several samples for four genes (LRRC3B, THRB, ITGA9 and RBSP3 (CTDSPL)) were bisulfite sequenced and confirmed the results of NMA hybridization. A set of eight biomarkers: NKIRAS1/RPL15, THRB, RBPS3 (CTDSPL), IQSEC1, NBEAL2, ZIC4, LOC285205 and FOXP1, was identified as the most prominent set capable to detect both early and late stages of ovarian cancer. Sensitivity of this set is equal to (72 ± 11)% and specificity (94 ± 5)%. Early stages represented the most complicated cases for detection. To distinguish between Stages I + II and Stages III + IV of ovarian cancer the most perspective set of biomarkers would include LOC285205, CGGBP1, EPHB1 and NKIRAS1/RPL15. The sensitivity of the set is equal to (80 ± 13)% and the specificity is (88 ± 12)%. Using this technique we plan to validate this panel with new epithelial ovarian cancer samples and add markers from other chromosomes.     

Hyaluronan Synthase and Hyaluronidase Expression in Serous Ovarian Carcinoma is Related to Anatomic Site and Chemotherapy Exposure

The present study investigated the expression and clinical role of hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-3) in serous ovarian carcinoma. HAS and HYAL mRNA expression was analyzed in 97 tumors (61 effusions, 27 primary carcinomas, 9 solid metastases) using PCR and further studied for association with clinicopathologic parameters, including survival. HAS1 mRNA was overexpressed in effusions compared to primary carcinomas and solid metastases (p < 0.001), and an alternatively spliced HAS1 was expressed only in effusions. HAS2 mRNA was overexpressed in solid metastases and primary carcinomas compared to effusions (p = 0.043), and HAS3 mRNA was overexpressed in primary carcinomas and effusions compared to solid metastases (p = 0.008). HYAL1 mRNA was absent in all specimens, whereas HYAL2 was expressed as two splice variants, of which HYAL2-var2 was overexpressed in solid metastases compared to effusions and primary carcinomas (p < 0.001). HYAL3 mRNA was expressed as wild-type and variant 1–3 form, the latter more highly in primary carcinomas and effusions compared to solid metastases (p = 0.006). HAS1 mRNA was overexpressed in pre- compared to post-chemotherapy effusions (p < 0.001), with opposite finding for HYAL2-var1 and HYAL3-WT (p = 0.016 and p = 0.024, respectively). Higher HYAL2-var1 and HAS1 splice variant mRNA expression in effusions was associated with longer (p = 0.033) and shorter (p = 0.047) overall survival, respectively. These data are the first to document a role for HAS and Hyal members in tumor progression in ovarian carcinoma, as evidenced by their differential expression as function of anatomic site and chemotherapy exposure, with a possible prognostic role for patients with malignant effusions.     

Expression Patterns and Functional Novelty of Ribonuclease 1 in Herbivorous Megalobrama amblycephala

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing.     

CYP24A1 Expression Inversely Correlates with Melanoma Progression: Clinic-Pathological Studies

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.     

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.     

Phenotypic and Molecular Convergence of 2q23.1 Deletion Syndrome with Other Neurodevelopmental Syndromes Associated with Autism Spectrum Disorder

Roughly 20% of autism spectrum disorders (ASD) are syndromic with a well-established genetic cause. Studying the genes involved can provide insight into the molecular and cellular mechanisms of ASD. 2q23.1 deletion syndrome (causative gene, MBD5) is a recently identified genetic neurodevelopmental disorder associated with ASD. Mutations in MBD5 have been found in ASD cohorts. In this study, we provide a phenotypic update on the prevalent features of 2q23.1 deletion syndrome, which include severe intellectual disability, seizures, significant speech impairment, sleep disturbance, and autistic-like behavioral problems. Next, we examined the phenotypic, molecular, and network/pathway relationships between nine neurodevelopmental disorders associated with ASD: 2q23.1 deletion Rett, Angelman, Pitt-Hopkins, 2q23.1 duplication, 5q14.3 deletion, Kleefstra, Kabuki make-up, and Smith-Magenis syndromes. We show phenotypic overlaps consisting of intellectual disability, speech delay, seizures, sleep disturbance, hypotonia, and autistic-like behaviors. Molecularly, MBD5 possibly regulates the expression of UBE3A, TCF4, MEF2C, EHMT1 and RAI1. Network analysis reveals that there could be indirect protein interactions, further implicating function for these genes in common pathways. Further, we show that when MBD5 and RAI1 are haploinsufficient, they perturb several common pathways that are linked to neuronal and behavioral development. These findings support further investigations into the molecular and pathway relationships among genes linked to neurodevelopmental disorders and ASD, which will hopefully lead to common points of regulation that may be targeted toward therapeutic intervention.