Neoplastic Transformation Of Epithelial Cells Research Articles

The Role of the Microbiome in the Etiopathogenesis of Colon Cancer.

Studies in preclinical models support that the gut microbiota play a critical role in the development and progression of colorectal cancer (CRC). Specific microbial species and their corresponding virulence factors or associated small molecules can contribute to CRC development and progression either via direct effects on the neoplastic transformation of epithelial cells or through interactions with the host immune system. Induction of DNA damage, activation of Wnt/β-catenin and NF-κB proinflammatory pathways, and alteration of the nutrient's availability and the metabolic activity of cancer cells are the main mechanisms by which the microbiota contribute to CRC. Within the tumor microenvironment, the gut microbiota alter the recruitment, activation, and function of various immune cells, such as T cells, macrophages, and dendritic cells. Additionally, the microbiota shape the function and composition of cancer-associated fibroblasts and extracellular matrix components, fashioning an immunosuppressive and pro-tumorigenic niche for CRC. Understanding the complex interplay between gut microbiota and tumorigenesis can provide therapeutic opportunities for the prevention and treatment of CRC.

IMMUNOHISTOCHEMICAL MARKERS IN DIAGNOSTICS OF ORAL PRECANCEROUS DISEASES

Over the past 15 years, the incidence of cancer of the oral cavity, pharynx, and larynx has increased by 15-17%, and almost 90% of patients are working people (30-60 years old). There were 32 patients (15 men and 17 women) aged 35 to 60 years with oral precancerous diseases examined at the department of Therapeutic Dentistry of Tashkent State Dental Institute. An IHC study in patients diagnosed with flat leukoplakia did not reveal neoplastic transformation of epithelial cells; the main changes in the epithelium were characterized by hyperplasia with cell proliferation.

Identification of KRAS gene codon 12 polymorphism in colorectal cancer patients at Mohammad Hoesin General Hospital Palembang

Colorectal cancer occurs due to neoplastic transformation of epithelial cells in the colon and rectum resulting from the accumulation of genetic and epigenetic aberrations. One of the genetic factors associated with colorectal cancer is KRAS gene polymorphism, which mostly occurs in codon 12. KRAS gene polymorphism leads to constitutional activation of KRAS protein effectors which will initiate signalling network to nucleus to increase cell proliferation, survival and differentiation. The aim of this study was to identify the KRAS gene codon 12 polymorphism in colorectal cancer patients at Mohammad Hoesin General Hospital Palembang. This study was a descriptive laboratory study with PCR–RFLP technique using BstNI enzyme on thirty patients with colorectal cancer. Wild type genotype (GG) of KRAS gene codon 12 found in 15 patients (50%), heterozygous mutant genotype (Gg) found in 12 patients (40%) and homozygous mutant genotype (gg) only found in 3 patients (10%). The G allele frequency was 70% and the g was 30%. The most common genotype found in colorectal cancer patients was GG genotype and most common alotype was G allele. KRAS gene polymorphism was found in half of the colorectal cancer patients, heterozygous mutant genotype (Gg) was more frequent then homozygous mutant genotype (gg).

Full-length genome sequence analysis of enzootic nasal tumor virus isolated from goats in China

BackgroundEnzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in China.MethodsTo evaluate the genetic diversity of ENTV-2 from Shaanxi province of China, the complete genome sequence of four isolates from Shaanxi province was determined by RT-PCR. These sequences were analyzed to evaluate their genetic relatedness with other small ruminant betaretroviruses. Phylogenetic analyses based on the gag gene and env gene were performed.ResultsThe ENTV-2-Shaanxi1 genome shared 97.0% sequence identity with ENTV-2-SC (accession number HM104174.1), and 89.6% sequence identity with the ENTV-2 sequences (accession number AY197548.1). ENTV-2 is closely related to the ENTV-1 and jaagsiekte retrovirus (JSRV). The main sequence differences between these viruses reside in LTR, two small regions of Gag, Orf-x, and the transmembrane (TM) region of Env. A stretch of 6 consecutive proline residues exists in VR1 of the ENTV-2-Shaanxi1 ~ 4 isolates. All the ENTV-2-Shaanxi isolates have the YXXM motif in the cytoplasmic tail of the Env. Phylogenetic analysis by nucleotide sequences showed that ENTV-2-Shaanxi1 ~ 4 isolates were closest related to two ENTV-2 isolates published in NCBI, especially with ENTV-2-SC strain.ConclusionsThis finding indicates that ENA most likely was introduced to Shaanxi province by the movement of contaminated goats from other areas in China. This study adds to understand the circulation, variation and distribution of ENTV-2, and may prove beneficial in future control or eradication programmes.

Can Targeting Stroma Pave the Way to Enhanced Antitumor Immunity and Immunotherapy of Solid Tumors?

Solid tumors are complex organ-like structures. The potential of normal neighboring cells to contribute to the initiation, progression, and metastasis of epithelial-derived carcinomas has long been appreciated. However, the role of host cells has proven complex. Through multiple local and systemic mechanisms, nontransformed host cells can promote transition from a tumor-resistant to tumor-permissive environment, drive neoplastic transformation of epithelial cells, promote tumor growth, progression, and metastasis, but also constrain tumorigenesis. This complexity reflects the spatially and temporally dynamic involvement of multiple cell types and processes, including the development and recruitment of inflammatory, immune, endothelial, and mesenchymal stromal cells, and the remodeling of extracellular matrix. Our mechanistic understanding, as well as our ability to translate advances in our understanding of these mechanisms for therapeutic benefit, is rapidly advancing. Further insights will depend on delineating pathways that mediate the communication networks between inflammatory and immune cells with tumor and mesenchymal stromal cells and extracellular matrix. Here, we discuss the diversity of mesenchymal stromal cell populations and how context can dictate either their promotion or constraint of tumorigenesis. We review evidence for plasticity that allows for reprogramming of stromal cells and how tumor immunogenicity and desmoplasia influence the balance of immune-independent and immune-dependent regulation of tumor growth. The pivotal roles of matrix and mesenchymal stromal cells in modulating inflammation, antitumor immunity, and the efficacy of immune-based therapies are discussed. These concepts have emerged from data obtained from tumors of multiple organs, but we focus mostly on studies of pancreatic ductal adenocarcinomas.

Epstein-Barr virus infection and nasopharyngeal carcinoma: the other side of the coin.

Oncogenic viruses may have a significant impact on the therapeutic management of several malignancies besides their well-known role in tumor pathogenesis. Epstein-Barr virus (EBV) induces neoplastic transformation of epithelial cells of the nasopharynx by various molecular mechanisms mostly involving activation of oncogenes and inactivation of tumor-suppressor genes. EBV infection can also induce the expression of several immunogenic peptides on the plasma membrane of the infected cells. Importantly, these virus-related antigens may be used as targets for antitumor immunotherapy-based treatment strategies. Two different immunotherapy strategies, namely adoptive and active immunotherapy, have been developed and strongly improved in the recent years. Furthermore, EBV infection may influence the use of targeted therapies for nasopharyngeal carcinoma (NPC) considering that the presence of EBV can induce important modifications in cell signaling. As an example, latent membrane protein type 1 is a viral transmembrane protein mainly involved in the cancerogenesis process, which can also mediate overexpression of the epidermal growth factor receptor (EGFR) in NPC cells, rendering them more sensitive to anti-EGFR therapy. Finally, EBV may induce epigenetic changes in the infected cells, such as DNA hypermethylation and histone deacetylation, that can sustain tumor growth and can thus be considered potential targets for novel therapies. In conclusion, EBV infection can modify important biological features of NPC cells, rendering them more vulnerable to both immunotherapy and targeted therapy.

Remodeling of angiogenesis and lymphangiogenesis in cervical cancer development

Ability to stimulate angiogenesis/lymphangiogenesis is recognized as an inherent feature of cancer cells providing necessary conditions for their growth and dissemination. "Angiogenic switch" is one of the earliest consequences of malignant transformation that encompasses a great number of genes and triggers a complex set of signaling cascades in endothelial cells. The processes of tumor microvasculature development are closely connected to the steps of carcinogenesis (from benign lesions to invasive forms) and occur through multiple deviations from the norm. Analysis of expression of proangiogenic factors at successive steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) enables to reconstruct the regulatory mechanisms of (lymph-)angiogenesis and to discriminate the most important components. This review presents detailed analysis of literature data on expression of the key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer. Their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis is discussed. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered, the potential of their use in molecular diagnostics and targeted therapy of cervical cancer is reviewed. Particular attention is paid to relatively poorly studied regulators of lymphangiogenesis and "non-VEGF dependent", or alternative, angiogenic pathways that constitute the prospect of future research in the field.

DRO1 inactivation drives colorectal carcinogenesis in ApcMin/+ mice.

Colorectal cancer develops from adenomatous precursor lesions by a multistep process that involves several independent mutational events in oncogenes and tumor suppressor genes. Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene is an early event and a prerequisite for the development of human colorectal adenoma. Previous in vitro studies identified DRO1 (CCDC80) to be a putative tumor suppressor gene that is negatively regulated in colorectal cancers and downregulated upon neoplastic transformation of epithelial cells. To investigate the in vivo role of DRO1 in colorectal carcinogenesis, a constitutive DRO1 knockout mouse model was generated. Disruption of DRO1 did not result in spontaneous intestinal tumor formation, consistent with the notion that DRO1 might have a role in suppressing the development of colon tumors in Apc(Min) (/+) mice, a widely used model for studying the role of APC in intestinal tumorigenesis that is hampered by the fact that mice predominantly develop adenomas in the small intestine and not in the colon. Here, deletion of DRO1 in Apc(Min) (/+) mice results in earlier death, a dramatically increased colonic tumor burden, and frequent development of colorectal carcinoma. Furthermore, enhanced phosphorylation of ERK1/2 is observed in colon epithelium and tumors from DRO1 knockout mice. Thus, this study reveals that inactivation of DRO1 is required for colorectal carcinogenesis in the Apc(Min) (/+) mouse and establishes a new mouse model for the study of colorectal cancer. This report characterizes a new mouse model for the study of colorectal cancer and establishes DRO1 (CCDC80) as a tumor suppressor via a mechanism involving ERK phosphorylation.

Abstract 1144: Cell plasticity mediated by EMT-inducing transcription factors contributes to melanoma development

Abstract Background: Embryonic transcription factors inducers of Epithelial to Mesenchymal Transition (EMT-TFs) are frequently reactivated during tumorigenesis. In addition to promoting metastasis in carcinoma, they favor neoplastic transformation of epithelial cells by enabling escape from oncogene-induced senescence and apoptosis and providing cells with stem-like properties. We recently unveiled different regulation and function of EMT-TFs in melanoma, a highly metastatic neural crest-derived cancer. We observed a switch in expression from SNAIL2 and ZEB2, which are expressed in melanocytes and behave as oncosuppressive proteins to TWIST1 and ZEB1, which are aberrantly reactivated in melanoma and cooperate with BRAFV600E oncogene to induce neoplastic transformation of melanocytes. This switch in EMT-TF expression represents a novel independent factor of poor prognosis in patients with malignant melanoma (Caramel et al, Cancer Cell, 2013). We further investigated the oncogenic properties of TWIST1 and ZEB1 in melanoma development and analyzed the crosstalk with the master regulator of melanoma phenotypic plasticity, the microphthalmia-associated transcription factor MITF. Methods: We first analysed the expression of MITF and TWIST1/ZEB1 in melanoma cell lines and human melanoma specimens by immunohistochemistry. We then studied the role of TWIST1 in melanoma progression in vivo by crossing conditional Twist1 transgenic mice with BRAFV600E/TyrCreERT2 mice. Results: Our data suggest that EMT-TFs control melanoma cell plasticity by modulating MITF level and the associated pathways. High MITF levels maintain the differentiated status of melanocytes, while reduction of MITF expression results in the transition to proliferative and invasive/stem states. TWIST1 and ZEB1 cooperate with BRAF in downregulating MITF concomitantly with the induction of invasion-associated gene signatures. A correlation of MITF expression with TWIST1 and ZEB1 is observed in human melanoma specimens at the intratumoral level. Moreover, while Twist1 enables BRAFV600E-induced senescence escape in primary mouse melanocytes, monitoring of the development of nevi and melanomas in the BRAFV600E mouse model showed more aggressive features in BRAFV600E/Twist1 mice compared to BRAFV600E control mice. Conclusion: Collectively, these data highlight crucial roles for TWIST1 and ZEB1 in promoting cell dedifferentiation and plasticity of non-epithelial cells. By regulating MITF-dependent phenotype switching they contribute to malignant progression of melanoma. Therefore, targeting the EMT-TF network represents an attractive strategy for metastatic melanomas that invariably develop resistance to BRAFV600 targeted therapy. Citation Format: Julie Caramel, Geoffrey Richard, Michelle Houang, Arnaud de la Fouchardiere, Lionel Larue, Richard Marais, Stephane Dalle, Eugene Tulchinsky, Stephane Ansieau, Alain Puisieux. Cell plasticity mediated by EMT-inducing transcription factors contributes to melanoma development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1144. doi:10.1158/1538-7445.AM2014-1144

Mesothelin expression in pancreatic mucinous cysts.

Mesothelin (MSLN) is a differentiation antigen found to be overexpressed in intraductal papillary mucinous neoplasms (IPMNs) and is a potential treatment target in pancreatic ductal adenocarcinoma. From institutional archives, 114 cases of resected pancreatic mucinous cysts were identified, including IPMN and mucinous cystic neoplasm (MCN). Immunohistochemical analysis of MSLN was performed on representative sections. MSLN was seen more frequently in neoplastic epithelial cells from IPMN (39/52; P < .0005) and MCN (9/14; P < .0001) compared with unremarkable adjacent pancreatic and bile ducts (0/57) and benign foveolar and duodenal epithelium (0/21). When present, MSLN was diffusely expressed in neoplastic epithelium and only focally expressed in adjacent ducts (8/57). No significant difference was seen (P = .26) in MLSN expression between IPMN (79%) and MCN (83%) when only presence or absence was considered. Our findings suggest that MLSN can be used as a marker of neoplastic transformation of epithelial cells in pancreatic mucinous cysts. The findings can help identify neoplastic mucinous epithelium.

Antagonistic functions of EMT-inducers in melanoma development: implications for cancer cell plasticity

EMT-inducing transcription factors (EMT-TFs) of the SNAIL, TWIST and ZEB families are frequently reactivated during tumorigenesis. In carcinoma, in addition to promoting metastasis, they favor neoplastic transformation of epithelial cells by enabling escape from failsafe programs and providing cells with stem-like properties. We recently unveiled different functions and regulatory mechanisms of EMT-TFs in melanoma, a highly metastatic neural crest-derived cancer. A switch in expression is observed from SNAIL2 and ZEB2, which are expressed in melanocytes and behave as oncosuppressive proteins to TWIST1 and ZEB1, which are aberrantly reactivated in melanoma and foster neoplastic transformation of melanocytes. This reversible switch in EMT-TF expression driven by a MEK-ERK-FRA1 pathway represents a novel independent factor of poor prognosis in patients with malignant melanoma. We herein discuss cell type specific biological functions of EMT-TFs in terms of cancer cell plasticity.

Cysteinyl leukotrienes and their receptors: Bridging inflammation and colorectal cancer

Long-standing inflammation has emerged as a hallmark of neoplastic transformation of epithelial cells and may be a limiting factor of successful conventional tumor therapies. A complex milieu composed of distinct stromal and immune cells, soluble factors and inflammatory mediators plays a crucial role in supporting and promoting various types of cancers. An augmented inflammatory response can predispose a patient to colorectal cancer (CRC). Common risk factors associated with CRC development include diet and lifestyle, altered intestinal microbiota and commensals, and chronic inflammatory bowel diseases. Cysteinyl leukotrienes are potent inflammatory metabolites synthesized from arachidonic acid and have a broad range of functions involved in the etiology of various pathologies. This review discusses the important role of cysteinyl leukotriene signaling in linking inflammation and CRC.

Modeling Tumor Progression by the Sequential Introduction of Genetic Alterations into the Genome of Human Normal Cells

Cancer genomes display a complex blend of genetic lesions affecting oncogenes and tumor suppressor genes. Multiple modeling approaches indicate that 5-15 driver oncogenic events are required to achieve tumor progression in common epithelial cancers. In vitro, a lower number (2-3) of events is typically sufficient to achieve full transformation. We developed cellular models that closely resemble the occurrence of multiple genetic lesions to understand their role in tumor progression. Homologous recombination and transcriptional downregulation were used to recapitulate the co-occurrence of driver mutations targeting oncogenes and inactivation of tumor suppressor genes in human nontransformed epithelial cells. Knockdown of the tumor suppressor genes PTEN or RB1 was combined with mutagenic activation of individual oncogenes (EGFR, KRAS, BRAF, or PIK3CA), thus generating a combinatorial model. The simultaneous presence of oncogenic and tumor suppressive events resulted in distinct biochemical properties and anchorage-independent growth abilities. Notably, however, we found that even when up to four individual alterations were concomitantly present they were not sufficient to fully transform the target cells. Our results suggest that the close recapitulation of cancer lesions in not-transformed cells is essential to unveil their oncogenic potential and raise questions concerning the minimal requirements for neoplastic transformation of epithelial cells.

CRC—all about microbial products and barrier function?

Novel findings draw attention to the adverse effect that neoplastic transformation of epithelial cells has on intestinal barrier function and cancer progression. An impaired intestinal barrier facilitated the translocation of microbial products, which promoted cytokine production and subsequent tumour progression in mice. The findings could open new therapeutic avenues for patients with early stage colorectal cancer.

Critical roles of the cAMP‐responsive element‐binding protein‐mediated pathway in disorganized epithelial phenotypes caused by mitochondrial dysfunction

In most human cancers, somatic mutations have been identified in the mtDNA; however, their significance remains unclear. We recently discovered that NMuMG mouse mammary epithelial cells, when deprived of mitochondria or following inhibition of respiratory activity, undergo epithelial morphological disruption accompanied with irregular edging of E-cadherin, the appearance of actin stress fibers, and an altered gene expression profile. In this study, using the mtDNA-less pseudo ρ0 cells obtained from NMuMG mouse mammary epithelial cells, we examined the roles of two mitochondrial stress-associated transcription factors, cAMP-responsive element-binding protein (CREB) and C/EBP homologous protein-10 (CHOP), in the disorganization of epithelial phenotypes. We found that the expression of matrix metalloproteinase-13 and that of GADD45A, SNAIL and integrin α1 in the ρ0 cells were regulated by CHOP and CREB, respectively. Of note, knockdown and pharmacological inhibition of CREB ameliorated the disrupted epithelial morphology. It is interesting to note that the expression of high mobility group AT-hook 2 (HMGA2), a non-histone chromatin protein implicated in malignant neoplasms, was increased at the protein level through the CREB pathway. Here, we reveal how the activation of the CREB/HMGA2 pathway is implicated in the repression of integrin α1 expression in HepG2 human cancer cells, highlighting the importance of the CREB/HMGA2 pathway in malignant transformation associated with mitochondrial dysfunction, thereby raising the possibility that the pathway indirectly interferes with the cell-cell adhesion structure by influencing the cell-extracellular matrix adhesion status. Overall, the data suggest that mitochondrial dysfunction potentially contributes to neoplastic transformation of epithelial cells through the activation of these transcriptional pathways.

Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1 NA) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1 EU). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1 EU genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1 NA and ENTV-1 EU reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K a/ K s ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.

Colorectal cancer: Potential therapeutic benefits of Vitamin D

Colorectal cancer is a disease that originates from the neoplastic transformation of epithelial cells of the colon and rectum, as a result of the accumulation of genetic and epigenetic aberrations. At least four sequential genetic changes, affecting one oncogen (KRAS) and three tumor suppressor genes (APC, SMAD4 and TP53), are required for the development of colorectal cancer. Abundant experimental studies and epidemiological data, as well as several human clinical trials suggest a protective effect of Vitamin D against colon carcinogenesis. Hypercalcemia, a side effect of natural Vitamin D, has currently restricted its therapeutic use; however, the development of new synthetic analogs with reduced hypercalcemic activity is promising for cancer therapy and prevention. Extensive research to elucidate the mechanisms underlying the anti-cancer action of Vitamin D is being undertaken. Understanding the complex molecular and cellular networks induced by Vitamin D or its analogs will improve the use of these compounds for the prevention and treatment of colorectal cancer.

Genomic patterns of allelic imbalance in disease free tissue adjacent to primary breast carcinomas

Mammary stroma plays an important role in facilitating the neoplastic transformation of epithelial cells, modulating integrity of the extracellular matrix, and maintaining genomic stability, but molecular mechanisms by which stroma affects epithelial structure and function are not well-defined. We used laser-assisted microdissection of paraffin-embedded breast tissues from 30 patients with breast disease and a panel of 52 microsatellite markers defining 26 chromosomal regions to characterize genomic patterns of allelic imbalance (AI) in disease-free tissue adjacent to sites of breast disease and to define genomic regions that may contain genes associated with early carcinogenic processes. The mean frequency of AI in histologically normal tissue adjacent to the primary carcinomas (15.4%) was significantly higher than that in distant tissue from the same breast (3.7%). The pattern of AI across all chromosomal regions differed between the adjacent tissue and primary tumor in every case. Unique AI events, observed only in tumor (15% of informative markers) or only in adjacent cells (10% of informative markers), were far more common than AI events shared between tumor and adjacent cells (approximately 4%). Levels of AI characteristic of advanced invasive carcinomas were already present in non-invasive ductal carcinomas in situ, and appreciable levels of AI were observed in adjacent non-neoplastic tissue at all pathological stages. Chromosome 11p15.1 showed significantly higher levels of AI in adjacent cells (p < 0.01), suggesting that this region may harbor genes involved in breast cancer development and progression. Our data indicate that genomic instability may be inherently greater in disease-free tissue close to developing tumors, which may have important implications for defining surgical margins and predicting recurrence.

Regulation of the Discs Large Tumor Suppressor by a Phosphorylation-dependent Interaction with the β-TrCP Ubiquitin Ligase Receptor

The discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.

Altered mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas.

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.