BACKGROUND

Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions.

METHODS

CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling.

RESULTS

Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8).

CONCLUSIONS

Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC.

BACKGROUND

Pheochromocytoma and paraganglioma are rare neural-crest-derived tumors. They are metastatic in 15% of cases, and the identification of a germline mutation in the SDHB gene is a predictive risk factor for malignancy and poor prognosis. To date, the link between SDHB mutations and malignancy is still missing.

OBJECTIVE

Epithelial to mesenchymal transition (EMT) is a developmental event, reactivated in cancer cells to promote cell mobility and invasiveness. The aim of this study was to address the participation of EMT in the metastatic evolution of pheochromocytoma/paraganglioma.

METHODS

Transcriptomic profiling of EMT was performed on 188 tumor samples, using a set of 94 genes implicated in this pathway. Activation of EMT was further confirmed at protein level by immunohistochemistry in a second set of 93 tumors.

RESULTS

Hierarchical unsupervised classification showed that most SDHB-metastatic samples clustered together, indicating that EMT is differently regulated in these tumors. Major actors of EMT, metalloproteases and components of cellular junctions, were either up-regulated (LOXL2, TWIST, TCF3, MMP2, and MMP1) or down-regulated (KRT19 and CDH2) in SDHB-metastatic tumors compared with nonmetastatic ones. Interestingly, within metastatic tumors, most of these genes (LOXL2, TWIST, TCF3, MMP2, and KRT19) also allowed us to discriminate SDHB-mutated from non-SDHB-related tumors. In the second set of tumors, we studied Snail1/2 expression by immunohistochemistry and observed its specific nuclear translocation in all SDHB-metastatic tumors.

CONCLUSIONS

We have identified the first pathway that distinguishes SDHB-metastatic from all other types of pheochromocytomas/paragangliomas and suggest that activation of the EMT process might play a critical role in the particularly invasive phenotype of this group of tumors.

The radial glial cells serve as neural progenitors and as a migratory guide for newborn neurons in the developing cerebral cortex. These functions require appropriate organization and proliferation of the polarized radial glial scaffold. Here, we demonstrate in mice that the myristoylated alanine-rich C-kinase substrate protein (MARCKS), a prominent cellular substrate for PKC, modulates radial glial placement and expansion. Loss of MARCKS results in ectopic collection of mitotically active radial progenitors away from the ventricular zone (VZ) in the upper cerebral wall. Apical restriction of key polarity complexes [CDC42, beta-catenin (CTNNB1), N-cadherin (CDH2), myosin IIB (MYOIIB), aPKCzeta, LGL, PAR3, pericentrin, PROM1] is lost. Furthermore, the radial glial scaffold in Marcks null cortex is compromised, with discontinuous, non-radial processes apparent throughout the cerebral wall and deformed, bulbous, unbranched end-feet at the basal ends. Further, the density of radial processes within the cerebral cortex is reduced. These deficits in radial glial development culminate in aberrant positioning of neurons and disrupted cortical lamination. Genetic rescue experiments demonstrate, surprisingly, that phosphorylation of MARCKS by PKC is not essential for the role of MARCKS in radial glial cell development. By contrast, the myristoylation domain of MARCKS needed for membrane association is essential for MARCKS function in radial glia. The membrane-associated targeting of MARCKS and the resultant polarized distribution of signaling complexes essential for apicobasal polarity may constitute a critical event in the appropriate placement, proliferation and organization of polarized radial glial scaffold in the developing cerebral cortex.

Throughout spermatogenesis, leptotene spermatocytes must traverse the blood-testis barrier (BTB) at stages VIII-XI to gain entry into the adluminal compartment for continued development. However, the mechanism underlying BTB restructuring remains somewhat elusive. In this study, interleukin 1 alpha (IL1A) was administered intratesticularly to adult rats in order to assess its effects on spermatogenesis. IL1A was shown to perturb Sertoli-germ cell adhesion, resulting in germ cell loss from approximately 50% of seminiferous tubules by 15 days posttreatment. Equally important, the functional integrity of the BTB was compromised when inulin-fluorescein isothiocyanate was detected in the adluminal compartment of the seminiferous epithelium following its administration via the jugular vein. Interestingly, IL1A did not affect the steady-state levels of proteins that confer BTB function, namely OCLN, CLDN1, F11R, TJP1, and CDH2. Instead, the localizations of OCLN, F11R, and TJP1 in the seminiferous epithelium were altered; these proteins appeared to move away from sites of cell-cell contact. Moreover, IL1A was shown to perturb the orderly arrangement of filamentous actin at the BTB and apical ectoplasmic specialization with distinct areas illustrating loss of actin filaments. Taken collectively, these results suggest that IL1A-induced BTB disruption is not mediated via the reduction of target protein levels. Instead, IL1A's primary cellular target appears to be the Sertoli cell actin cytoskeleton. It is possible that localized production of IL1A by Sertoli and/or germ cells in vivo results in BTB restructuring, and this may facilitate the movement of leptotene spermatocytes across the BTB.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5' regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5' regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription-PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5' CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.

BACKGROUND

Obsessive-compulsive disorder (OCD), a severe mental disease manifested in time-consuming repetition of behaviors, affects 1 to 3% of the human population. While highly heritable, complex genetics has hampered attempts to elucidate OCD etiology. Dogs suffer from naturally occurring compulsive disorders that closely model human OCD, manifested as an excessive repetition of normal canine behaviors that only partially responds to drug therapy. The limited diversity within dog breeds makes identifying underlying genetic factors easier.

RESULTS

We use genome-wide association of 87 Doberman Pinscher cases and 63 controls to identify genomic loci associated with OCD and sequence these regions in 8 affected dogs from high-risk breeds and 8 breed-matched controls. We find 119 variants in evolutionarily conserved sites that are specific to dogs with OCD. These case-only variants are significantly more common in high OCD risk breeds compared to breeds with no known psychiatric problems. Four genes, all with synaptic function, have the most case-only variation: neuronal cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). In the 2 Mb gene desert between the cadherin genes CDH2 and DSC3, we find two different variants found only in dogs with OCD that disrupt the same highly conserved regulatory element. These variants cause significant changes in gene expression in a human neuroblastoma cell line, likely due to disrupted transcription factor binding.

CONCLUSIONS

The limited genetic diversity of dog breeds facilitates identification of genes, functional variants and regulatory pathways underlying complex psychiatric disorders that are mechanistically similar in dogs and humans.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

Accumulating evidence suggests that the mechanical and biochemical signals originating from cell-cell adhesion are critical for stem cell lineage specification. In this review, we focus on the role of cadherin mediated signaling in development and stem cell differentiation, with emphasis on two well-known cadherins, cadherin-2 (CDH2) (N-cadherin) and cadherin-11 (CDH11) (OB-cadherin). We summarize the existing knowledge regarding the role of CDH2 and CDH11 during development and differentiation in vivo and in vitro. We also discuss engineering strategies to control stem cell fate decisions by fine-tuning the extent of cell-cell adhesion through surface chemistry and microtopology. These studies may be greatly facilitated by novel strategies that enable monitoring of stem cell specification in real time. We expect that better understanding of how intercellular adhesion signaling affects lineage specification may impact biomaterial and scaffold design to control stem cell fate decisions in three-dimensional context with potential implications for tissue engineering and regenerative medicine.

The epithelial-to-mesenchymal (-like) transition (EMT), a crucial embryonic development program, has been linked to the regulation of glioblastoma (GBM) progression and invasion. Here, we investigated the role of MIR517C/miR-517c, which belongs to the C19MC microRNA cluster identified in our preliminary studies, in the pathogenesis of GBM. We found that MIR517C was associated with improved prognosis in patients with GBM. Furthermore, following treatment with the autophagy inducer temozolomide (TMZ) and low glucose (LG), MIR517C degraded KPNA2 (karyopherin alpha 2 [RAG cohort 1, importin alpha 1]) and subsequently disturbed the nuclear translocation of TP53 in the GBM cell line U87 in vitro. Interestingly, this microRNA could inhibit autophagy and reduce cell migration and infiltration in U87 cells harboring wild-type (WT) TP53, but not in U251 cells harboring mutant (MU) TP53. Moreover, the expression of epithelial markers (i.e., CDH13/T-cadherin and CLDN1 [claudin 1]) increased, while the expression of mesenchymal markers (i.e., CDH2/N-cadherin, SNAI1/Snail, and VIM [vimentin]) decreased, indicating that the EMT status was blocked by MIR517C in U87 cells. Compared with MIR517C overexpression, MIR517C knockdown promoted infiltration of U87 cells to the surrounding structures in nude mice in vivo. The above phenotypic changes were also observed in TP53(+/+) and TP53(-/-) HCT116 colon cancer cells. In summary, our study provided support for a link between autophagy and EMT status in WT TP53 GBM cells and provided evidence for the signaling pathway (MIR517C-KPNA2-cytoplasmic TP53) involved in attenuating autophagy and eliminating the increased migration and invasion during the EMT.

Studies have shown that miR-194 functions as a tumor suppressor and is associated with tumor growth and metastasis. We studied the effects of miR-194 in osteosarcoma and the possible mechanism by which miR-194 affected the survival, apoptosis and metastasis of osteosarcoma. Both human osteosarcoma cell lines SOSP-9607 and U2-OS were transfected with recombinant lentiviruses to regulate miR-194 expression. Overexpression of miR-194 partially inhibited the proliferation, migration, and invasion of osteosarcoma cells in vitro, as well as tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. Potential miR-194 target genes were predicted using bioinformatics. Luciferase reporter assay, real-time quantitative PCR and western blotting confirmed that CDH2 (N-cadherin) and IGF1R were targets of miR-194. Using real-time quantitative PCR, we evaluated the expression of miR-194 and two miR-194 target genes, CDH2 and IGF1R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed paraffin-embedded tissues. The expressions of the target genes were also examined in osteosarcoma samples using immunohistochemistry. Overexpression of miR-194 inhibited tumor growth and metastasis of osteosarcoma probably by downregulating CDH2 and IGF1R. miR-194 may prove to be a promising therapeutic agent for osteosarcoma.

Leiomyosarcoma is one of the most common mesenchymal tumors. Proteomics profiling analysis by reverse-phase protein lysate array surprisingly revealed that expression of the epithelial marker E-cadherin (encoded by CDH1) was significantly elevated in a subset of leiomyosarcomas. In contrast, E-cadherin was rarely expressed in the gastrointestinal stromal tumors, another major mesenchymal tumor type. We further sought to 1) validate this finding, 2) determine whether there is a mesenchymal to epithelial reverting transition (MErT) in leiomyosarcoma, and if so 3) elucidate the regulatory mechanism responsible for this MErT. Our data showed that the epithelial cell markers E-cadherin, epithelial membrane antigen, cytokeratin AE1/AE3, and pan-cytokeratin were often detected immunohistochemically in leiomyosarcoma tumor cells on tissue microarray. Interestingly, the E-cadherin protein expression was correlated with better survival in leiomyosarcoma patients. Whole genome microarray was used for transcriptomics analysis, and the epithelial gene expression signature was also associated with better survival. Bioinformatics analysis of transcriptome data showed an inverse correlation between E-cadherin and E-cadherin repressor Slug (SNAI2) expression in leiomyosarcoma, and this inverse correlation was validated on tissue microarray by immunohistochemical staining of E-cadherin and Slug. Knockdown of Slug expression in SK-LMS-1 leiomyosarcoma cells by siRNA significantly increased E-cadherin; decreased the mesenchymal markers vimentin and N-cadherin (encoded by CDH2); and significantly decreased cell proliferation, invasion, and migration. An increase in Slug expression by pCMV6-XL5-Slug transfection decreased E-cadherin and increased vimentin and N-cadherin. Thus, MErT, which is mediated through regulation of Slug, is a clinically significant phenotype in leiomyosarcoma.

Colorectal cancer (CRC) is the most common cancer diagnosed worldwide, and the development of metastases is a major cause of mortality. Accumulating evidence suggests that microRNAs are important in carcinogenesis by affecting the expression of genes that regulate cancer progression. A number of studies have shown that miR-206 is frequently downregulated in many human malignancies, including CRC, and is associated with a more malignant phenotype. Previous studies involving HeLa and C2C12 cells have validated the inhibitory mechanism of miR-206 via NOTCH3 targeting. However, whether or not the interplay between miR-206 and NOTCH3 also occurs in CRC is unknown. Therefore, we investigated the tumor suppressive and metastatic effects of miR-206 and its target, NOTCH3, in CRC. Based on the inverse association between the expression of miR-206 and NOTCH3 in CRC tissues, miR-206 mimics were transiently transfected into the SW480 (and its metastatic strain) and SW620 colon cancer cell lines. Upregulation of miR-206 inhibited cancer cell prolife-ration and migration, blocked the cell cycle, and activated apoptosis. The tumor suppressive capacity of miR-206 had a similar effect on CRC cells, although with a different metastatic potential, and may be explained by direct NOTCH3 signaling inhibition and indirect cross-talk with other signaling pathways involving CDH2 and MMP-9. These results support miR-206 as a tumor suppressor in CRC and suggest a potential therapeutic target for clinical intervention.

Pleural malignant mesothelioma (MM) is an aggressive cancer with a very long latency and a very short median survival. Little is known about the genetic events that trigger MM and their relation to poor outcome. The goal of our study was to characterize major genomic gains and losses associated with MM origin and progression and assess their clinical significance. We performed Representative Oligonucleotide Microarray Analysis (ROMA) on DNA isolated from tumors of 22 patients who recurred at variable interval with the disease after surgery. The total number of copy number alterations (CNA) and frequent imbalances for patients with short time (<12 months from surgery) and long time to recurrence were recorded and mapped using the Analysis of Copy Errors algorithm. We report a profound increase in CNA in the short-time recurrence group with most chromosomes affected, which can be explained by chromosomal instability associated with MM. Deletions in chromosomes 22q12.2, 19q13.32 and 17p13.1 appeared to be the most frequent events (55-74%) shared between MM patients followed by deletions in 1p, 9p, 9q, 4p, 3p and gains in 5p, 18q, 8q and 17q (23-55%). Deletions in 9p21.3 encompassing CDKN2A/ARF and CDKN2B were characterized as specific for the short-term recurrence group. Analysis of the minimal common areas of frequent gains and losses identified candidate genes that may be involved in different stages of MM: OSM (22q12.2), FUS1 and PL6 (3p21.3), DNAJA1 (9p21.1) and CDH2 (18q11.2-q12.3). Imbalances seen by ROMA were confirmed by Affymetrix genome analysis in a subset of samples.

Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies but the molecular genetic basis of this disease remains unclear. By using genome-wide methylation profiling analysis, we identified CLDN3 as an epigenetically regulated gene in cancer. Here, we investigated its function and clinical relevance in human HCC. CLDN3 downregulation occurred in 87/114 (76.3%) of primary HCCs, where it was correlated significantly with shorter survival of HCC patients (P=0.021). Moreover, multivariate cyclooxygenase regression analysis showed that CLDN3 was an independent prognostic factor for overall survival (P=0.014). Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation. Ectopic expression of CLDN3 in HCC cells could inhibit cell motility, cell invasiveness, and tumor formation in nude mice. Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells. Collectively, our findings demonstrated that CLDN3 is an epigenetically silenced metastasis suppressor gene in HCC. A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.

Identification and characterization of crucial gene target(s) that will allow focused therapeutics development remains a challenge. We have interrogated the putative therapeutic targets associated with the transcription factor Grainy head-like 2 (GRHL2), a critical epithelial regulatory factor. We demonstrate the possibility to define the molecular functions of critical genes in terms of their personalized expression profiles, allowing appropriate functional conclusions to be derived. A novel methodology, relative expression analysis with gene-set pairs (RXA-GSP), is designed to explore the potential clinical utility of cancer-biology discovery. Observing that Grhl2-overexpression leads to increased metastatic potential in vitro, we established a model assuming Grhl2-induced or -inhibited genes confer poor or favorable prognosis respectively for cancer metastasis. Training on public gene expression profiles of 995 breast cancer patients, this method prioritized one gene-set pair (GRHL2, CDH2, FN1, CITED2, MKI67 versus CTNNB1 and CTNNA3) from all 2717 possible gene-set pairs (GSPs). The identified GSP significantly dichotomized 295 independent patients for metastasis-free survival (log-rank tested p = 0.002; severe empirical p = 0.035). It also showed evidence of clinical prognostication in another independent 388 patients collected from three studies (log-rank tested p = 3.3e-6). This GSP is independent of most traditional prognostic indicators, and is only significantly associated with the histological grade of breast cancer (p = 0.0017), a GRHL2-associated clinical character (p = 6.8e-6, Spearman correlation), suggesting that this GSP is reflective of GRHL2-mediated events. Furthermore, a literature review indicates the therapeutic potential of the identified genes. This research demonstrates a novel strategy to integrate both biological experiments and clinical gene expression profiles for extracting and elucidating the genomic impact of a novel factor, GRHL2, and its associated gene-sets on the breast cancer prognosis. Importantly, the RXA-GSP method helps to individualize breast cancer treatment. It also has the potential to contribute considerably to basic biological investigation, clinical tools, and potential therapeutic targets.

Understanding the genetic basis of common disease and disease-related quantitative traits will aid in the development of diagnostics and therapeutics. The processs of gene discovery can be sped up by rapid and effective integration of well-defined mouse genome and phenome data resources. We describe here an in silico gene-discovery strategy through genome-wide association (GWA) scans in inbred mice with a wide range of genetic variation. We identified 937 quantitative trait loci (QTLs) from a survey of 173 mouse phenotypes, which include models of human disease (atherosclerosis, cardiovascular disease, cancer and obesity) as well as behavioral, hematological, immunological, metabolic, and neurological traits. 67% of QTLs were refined into genomic regions <0.5 Mb with approximately 40-fold increase in mapping precision as compared with classical linkage analysis. This makes for more efficient identification of the genes that underlie disease. We have identified two QTL genes, Adam12 and Cdh2, as causal genetic variants for atherogenic diet-induced obesity. Our findings demonstrate that GWA analysis in mice has the potential to resolve multiple tightly linked QTLs and achieve single-gene resolution. These high-resolution QTL data can serve as a primary resource for positional cloning and gene identification in the research community.

Over 200 rare and fully penetrant pathogenic mutations in amyloid precursor protein (APP), presenilin 1 and 2 (PSEN1 and PSEN2) cause a subset of early-onset familial Alzheimer's disease (EO-FAD). Of these, 21 cases of EO-FAD families carrying unique APP locus duplications remain the only pathogenic copy number variations (CNVs) identified to date in Alzheimer's disease (AD). Using high-density DNA microarrays, we performed a comprehensive genome-wide analysis for the presence of rare CNVs in 261 EO-FAD and early/mixed-onset pedigrees. Our analysis revealed 10 novel private CNVs in 10 EO-FAD families overlapping a set of genes that includes: A2BP1, ABAT, CDH2, CRMP1, DMRT1, EPHA5, EPHA6, ERMP1, EVC, EVC2, FLJ35024 and VLDLR. In addition, CNVs encompassing two known frontotemporal dementia genes, CHMP2B and MAPT were found. To our knowledge, this is the first study reporting rare gene-rich CNVs in EO-FAD and early/mixed-onset AD that are likely to underlie pathogenicity in familial AD and perhaps related dementias.

Recurrent gain on chromosome 3q26 encompassing the gene locus for the transcription factor SOX2 is a frequent event in human squamous cell carcinoma, including head and neck squamous cell carcinoma (HNSCC). Numerous studies demonstrated that SOX2 expression and function is related to distinct aspects of tumor cell pathophysiology. However, the underlying molecular mechanisms are not well understood, and the correlation between SOX2 expression and clinical outcome revealed conflicting data. Transcriptional profiling after silencing of SOX2 expression in a HNSCC cell line identified a set of up-regulated genes related to cell motility (e.g. VIM, FN1, CDH2). The inverse regulation of SOX2 and aforementioned genes was validated in 18 independent HNSCC cell lines from different anatomical sites. The inhibition of cell migration and invasion by SOX2 was confirmed by constant or conditional gene silencing and accelerated motility of HNSCC cells after SOX2 silencing was partially reverted by down-regulation of vimentin. In a retrospective study, SOX2 expression was determined by immunohistochemical staining on tissue microarrays containing primary tumor specimens of two independent HNSCC patient cohorts. Low SOX2 expression was found in 19.3% and 44.9% of primary tumor specimens, respectively. Univariate analysis demonstrated a statistically significant correlation between low SOX2 protein levels and reduced progression-free survival (Cohort I 51 vs. 16 months; Cohort II 33 vs. 12 months) and overall survival (Cohort I 150 vs. 37 months; Cohort II 33 vs. 16 months). Multivariate Cox proportional hazard model analysis confirmed that low SOX2 expression serves as an independent prognostic marker for HNSCC patients. We conclude that SOX2 inhibits tumor cell motility in HNSCC cells and that low SOX2 expression serves as a prognosticator to identify HNSCC patients at high risk for treatment failure.

During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the blood-testis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP, and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with adjudin [a molecule that induces adherens junction disruption, formerly called AF-2364, 1-(2,4-dichlorobenzyl)-IH-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, coimmunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the N-cadherin (CDH2)/beta-catenin (CATNB)/actin complex but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia tranlocation to 4 homolog, MLLT4) nor the integrin beta1 (ITB1)-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the adjudin-mediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NO signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. Perhaps the most important of all, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway.

BACKGROUND

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous condition caused by mutations in genes encoding desmosomal proteins in up to 60% of cases. The 40% of genotype-negative cases point to the need of identifying novel genetic substrates by studying genotype-negative ARVC families.

RESULTS

Whole exome sequencing was performed on 2 cousins with ARVC. Validation of 13 heterozygous variants that survived internal quality and frequency filters was performed by Sanger sequencing. These variants were also genotyped in all family members to establish genotype-phenotype cosegregation. High-resolution melting analysis followed by Sanger sequencing was used to screen for mutations in cadherin 2 (CDH2) gene in unrelated genotype-negative patients with ARVC. In a 3-generation family, we identified by whole exome sequencing a novel mutation in CDH2 (c.686A>C, p.Gln229Pro) that cosegregated with ARVC in affected family members. The CDH2 c.686A>C variant was not present in >200 000 chromosomes available through public databases, which changes a conserved amino acid of cadherin 2 protein and is supported as the causal mutation by parametric linkage analysis. We subsequently screened 73 genotype-negative ARVC probands tested previously for mutations in known ARVC genes and found an additional likely pathogenic variant in CDH2 (c.1219G>A, p.Asp407Asn). CDH2 encodes cadherin 2 (also known as N-cadherin), a protein that plays a vital role in cell adhesion, making it a biologically plausible candidate gene in ARVC pathogenesis.

CONCLUSIONS

These data implicate CDH2 mutations as novel genetic causes of ARVC and contribute to a more complete identification of disease genes involved in cardiomyopathy.

BACKGROUND

Down-regulation of E-cadherin (CDH1) and epithelial-mesenchymal transition (EMT) are considered critical events for invasion and metastasis of colorectal carcinoma. Here we tested whether the important regulators of E-cadherin expression SNAI1 and TWIST1 are already detectable in human colorectal adenomas.

METHODS

RNA was extracted from a set of randomly selected formalin-fixed and paraffin-embedded (FFPE) colorectal adenomas (n = 41) and normal colon mucosa (n = 10). Subsequently mRNA expression of CDH1, CDH2, SNAI1 and TWIST1 was analysed by quantitative RT-PCR analysis. CDH1 as well as SNAI1 protein expression were assessed by immunohistochemistry (IHC).

RESULTS

SNAI1 mRNA was expressed in 78% (n = 32/41), TWIST1 mRNA in 41% (n = 17/41) and CDH2 mRNA in 41% (n = 17/41) of the colorectal adenoma tissue, while normal colon mucosa was negative for these transcription factors. We found a significant correlation between reduced CDH1 and the presence of SNAI1 mRNA expression and for combined SNAI1 and TWIST1 mRNA expression, respectively. A correlation between CDH2 mRNA expression and reduced CDH1 expression was not observed. We confirmed the relationship between SNAI1 expression and reduced E-cadherin expression on the protein level via IHC.

CONCLUSIONS

Our data show that SNAI1 and Twist1 are already expressed in benign precursor lesions of colorectal cancer and that SNAI1 expression was significantly correlated with lower expression of CDH1. Whether these findings reflect true EMT and/or are a sign of a more aggressive biology need to be investigated in further studies.

OBJECTIVE

Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance.

METHODS

We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis.

RESULTS

The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma.

CONCLUSIONS

Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.

Ovariectomy-induced bone loss is accentuated in mice with germline Cdh2 haploinsufficiency, the result of a decreased osteoblastogenesis in the face of normal osteoclast number. Reduced N-cadherin abundance in these mice decreases cell-cell adhesion and alters signaling pathways important for osteoblast commitment and differentiation, thus providing in vivo evidence that N-cadherin-mediated cell-cell interactions are involved in homeostatic responses to increased bone remodeling.

BACKGROUND

We have shown that targeted expression of a dominant negative truncated form of N-cadherin (Cdh2) delays acquisition of peak bone mass in mice and retards osteoblast differentiation. We tested the role of this molecule in the skeletal homeostatic response to ovariectomy in mice with germline Cdh2 haploinsufficiency.

METHODS

Heterozygous Cdh2 null (Cdh2+/-) and wildtype mice were ovariectomized and followed up to 13 weeks by in vivo radiodensitometric and ex vivo histologic assessment of bone mass and turnover. Cells isolated from wildtype and Cdh2+/- mice were used to determine the alterations in bone cell function produced by partial loss of N-cadherin.

RESULTS

Bone mass was not significantly different between Cdh2+/- and wildtype littermates, but on ovariectomy, bone loss in Cdh2+/- mice was initially slower, but with time it became significantly greater than in wildtype mice. This accentuated bone loss was associated with lower osteoblast number and serum osteocalcin levels, with no differences in bone resorption. Although development of calcified nodules was faster in calvaria cells isolated from Cdh2+/- mice relative to Cdh2+/+ cells, bone marrow osteogenic precursors were lower in the former than in the latter genotypes. Cdh2 expression was downregulated with differentiation in wildtype calvaria cells, whereas cadherin-11 abundance remained unchanged. Furthermore, cell-cell adhesion (postconfluence) was decreased among heterozygous calvaria cells, as was cell proliferation (preconfluence), relative to wildtype cells. Finally, the abundance and cellular distribution of beta-catenin was minimally decreased in Cdh2+/- cells, whereas mitogen-activated protein kinase (MAPK) signaling was more active in Cdh2 insufficient cells.

CONCLUSIONS

Cdh2 is involved in the homeostatic bone formation response to ovariectomy, presumably by regulating osteoprogenitors number and differentiation through stabilization of cell-cell adhesion and/or signaling modulation.