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Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Many of the 20-member mucin family are evolutionarily conserved proteins that are often aberrantly expressed and glycosylated in various benign and malignant pathologies including oncogenic signaling leading to tumor invasion, metastasis, and immune evasion. Large size and extensive glycosylation present challenges to study mucin structure using traditional methods, including crystallography. We offer the hypothesis that the functional versatility of mucins may be attributed to the presence of intrinsically disordered regions (IDRs), which provide dynamism and flexibility; further, that these sites offer potential therapeutic targets. Herein, we examined the links between mucin structure and function based on IDRs, post-translational modifications (PTMs), and potential impact on their interactome. Using sequence-based bioinformatics tools, we observed that mucins are predicted to be moderately (20–40%) to highly (>40%) disordered and many conserved mucin domains could be disordered. Phosphorylation sites overlap with IDRs throughout the mucin sequences. Additionally, the majority of predicted O- and N- glycosylation sites in the tandem repeat regions occur within IDRs, and these IDRs contain a large number of functional motifs, i.e. molecular recognition features (MoRFs), which directly influence PPIs. This investigation provides a novel perspective and offers an insight into the complexity and dynamic nature of mucins.

The majority of pancreatic cancer (PC) patients are clinically presented with obstructive jaundice with elevated levels of circulatory bilirubin and alkaline phosphatases. In the current study, we examined the implications of bile acids (BA), an important component of bile, on the pathophysiology of PC and investigated their mechanistic association in tumor‐promoting functions. Integration of results from PC patient samples and autochthonous mouse models showed an elevated levels of BA (p

Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane‐mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β‐catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF‐binding sites, leading us to hypothesize that MUC4 can be regulated by β‐catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β‐catenin expression. Knock down (KD) of β‐catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β‐catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., −2629/−2612) and furthest from the start site (i.e., −3425/−3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β‐catenin on −2629/−2612 and −3425/−3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β‐catenin KD cells showed decreased migration and decreased Vimentin, N‐cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β‐catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β‐catenin directly governs MUC4 in PC.

BACKGROUND Prostate cancer (PCa) cells express Toll-like receptor-4 (TLR4), a known pro-tumorigenic molecule for different cancer cells. The cancer cells residing in the avascular region of the tumor confront various metabolic stresses and continuously adapt mechanisms to overcome them. We hypothesized that TLR4 activation might provide direct survival advantage to metabolically stressed PCa cells. METHODS We first investigated the effect of LPS on survival of serum deprived PCa cells. To understand the molecular mechanisms involved in TLR4 mediated PCa survival, we next investigated change in expression of markers for apoptosis, senescence and autophagy. Ultimately, the effect of LPS on established prostate tumors was confirmed in vivo using a syngeneic rat model for PCa. RESULTS Lipopolysaccharide (LPS)-mediated TLR4 activation significantly enhanced survival of serum deprived (SD) PC3, DU145 and MAT-LyLu PCa cells. TLR4 inhibition by a specific inhibitor resulted in rapid death of SD-PC3 cells, which was significantly suppressed by LPS. Interestingly, LPS treatment suppressed macroautophagy in SD-PC3 cells and increased expression of CCL2 (C–C motif ligand-2), a known autophagy inhibitor and pro-survival factor. Intra-tumor LPS injection resulted in increased tumor mass, induced TLR4 activation, suppressed autophagy, and increased the macrophage population in MAT-LyLu-tumors. CONCLUSIONS Our study reveals that bacterial LPS enhance survival of PCa cells under conditions of nutrient stress through TLR4 activation. Moreover, LPS induces overexpression of CCL2 involved in the suppression of starvation-induced macroautophagy in PCa cells, and enhanced macrophage population in prostate tumors in vivo. Taken together, the current study suggests the importance of bacterial infection or TLR4-activation in prostate cancer pathogenesis. Prostate 75:1020–1033, 2015. © 2015 Wiley Periodicals, Inc.

MUC16 is a high-molecular-weight glycoprotein that is expressed by the various epithelial cell surfaces of the human body to protect the cell layer from a myriad of insults. It is the largest mucin known to date, with an ∼22,152 aa sequence. Structurally, MUC16 is characterized into 3 distinct domains: the amino terminal, the tandem repeat, and the carboxyl terminal domain, with each domain having unique attributes. The extracellular portion of MUC16 is shed into the bloodstream and serves as a biomarker for diagnosing and monitoring patients with cancer; however, its functional role in cancer is yet to be elucidated. Several factors contribute to this challenge, which include the large protein size; the extensive glycosylation that the protein undergoes, which confers functional heterogeneity; lack of specific antibodies that detect the unique domains of MUC16; and the existence of splicing variants. Despite these limitations, MUC16 has been established as a molecule of significant application in cancer. Hence, in this review, we discuss the various aspects of MUC16, which include its discovery, structure, and biological significance both in benign and malignant conditions with an attempt to dissect its functional relevance

Aim People of Hispanic origin comprise nearly 16 percent of the (US) population. With the growing population of Hispanics in the USA, an important epidemiological question is whether their country of origin affects survival in Hispanic women living in the USA at the time of diagnosis of breast cancer.

Accumulating lines of experimental evidence have revealed that hypoxia-inducible factors, HIF-1α and HIF-2α, are key regulators of the adaptation of cancer- and metastasis-initiating cells and their differentiated progenies to oxygen and nutrient deprivation during cancer progression under normoxic and hypoxic conditions. Particularly, the sustained stimulation of epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), stem cell factor (SCF) receptor KIT, transforming growth factor-β receptors (TGF-βRs) and Notch and their downstream signalling elements such as phosphatidylinositol 3'-kinase (PI3K)/Akt/molecular target of rapamycin (mTOR) may lead to an enhanced activity of HIFs. Moreover, the up-regulation of HIFs in cancer cells may also occur in the hypoxic intratumoral regions formed within primary and secondary neoplasms as well as in leukaemic cells and metastatic prostate and breast cancer cells homing in the hypoxic endosteal niche of bone marrow. The activated HIFs may induce the expression of numerous gene products such as induced pluripotency-associated transcription factors (Oct-3/4, Nanog and Sox-2), glycolysis- and epithelial-mesenchymal transition (EMT) programme-associated molecules, including CXC chemokine receptor 4 (CXCR4), snail and twist, microRNAs and angiogenic factors such as vascular endothelial growth factor (VEGF). These gene products in turn can play critical roles for high self-renewal ability, survival, altered energy metabolism, invasion and metastases of cancer cells, angiogenic switch and treatment resistance. Consequently, the targeting of HIF signalling network and altered metabolic pathways represents new promising strategies to eradicate the total mass of cancer cells and improve the efficacy of current therapies against aggressive and metastatic cancers and prevent disease relapse.

BACKGROUND: DF3/MUC1 mucin is expressed in various cancer tissues, and many in vitro studies have suggested that it may play a role in the aggressive behavior of malignant tumors. However, to the best of the authors' knowledge, the relation between DF3/MUC1 expression and outcome has not yet been investigated in patients with oral squamous cell carcinoma (OSCC). The objective of the current study was to evaluate the prognostic significance of DF3/MUC1 expression in patients with OSCC. METHODS: The expression profile of DF3/MUC1 in OSCC tissues from 206 patients was examined using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed on the basis of detailed clinicopathologic factors. RESULTS: DF3/MUC1 expression was found to be significantly correlated with tumor aggressiveness, such as pathologic lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01). Respectively, the overall survival (OS)and disease-free survival (DFS) rates were significantly worse for patients with DF3/MUC1 expression compared with those without DF3/MUC1 expression (P = .001 and P = .0003, respectively). Multivariate analysis demonstrated that DF3/MUC1 expression was an independent prognostic factor for both OS and DFS (P = .04 for both). In addition, DF3/MUC1 expression was found to be an independent risk factor for subsequent regional lymph node metastasis (P = .03). CONCLUSIONS: Aberrant expression of DF3/MUC1 is an independent prognostic factor indicating poor prognosis in patients with OSCC. DF3/MUC1 expression is a risk factor for subsequent lymph node metastasis in patients with OSCC and therefore may represent an indication for elective neck dissection. Patients with OSCC demonstrating positive expression of DF3/MUC1 should be followed carefully. Cancer 2012. © 2012 American Cancer Society.

BACKGROUND: The authors previously reported that neutrophil gelatinase-associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)-EGF receptor (EGFR) axis is up-regulated significantly in PDAC, they examined EGF-mediated NGAL regulation in these cells. METHODS: The NGAL-positive cell lines AsPC-1 and BxPC-3 were used as a model system. Quantitative real-time polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or small hairpin RNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay. RESULTS: NGAL expression was positively associated with tumor differentiation and was down-regulated significantly after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin down-regulation was partly through the EGFR-dependent mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) (MEK-ERK) signaling pathway. In addition, E-cadherin down-regulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued the inhibition of NGAL expression by EGF. Furthermore, EGF, in part through E-cadherin, reduced NGAL promoter activity by blocking nuclear factor κB (NF-κB) activation. CONCLUSIONS: The current study demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect was mediated in part through activation of the EGFR-MEK-ERK signaling pathway, which, in turn, down-regulated E-cadherin with a subsequent reduction in NF-κB activation. These findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. Cancer 2011;. © 2010 American Cancer Society.

Aim: Only limited data are available regarding mucin antigen expression in adenocarcinoma of the uterine cervix. Methods: Fifty-two patients with mucinous adenocarcinoma of the endocervical type were enrolled for making clear implication of the mucin antigens. The expression of mucin antigens in hysterectomized specimens were determined immunohistochemically. Clinicopathological characteristics and prognosis included International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, nodal and ovarian metastases, overall and disease-free survivals. The relationships of the expression of mucin antigens to various variables were investigated using uni- and multivariate analyses. Results: The majority of mucinous adenocarcinoma showed overexpression of MUC1 and MUC16. Overexpression of both MUC1 and MUC16 was associated with lower survival rates. Particularly, overexpression of MUC1 was associated with a lower disease-free survival rate and lymph node metastasis. However, absence of expression of MUC1 and/or MUC16 was associated with longer overall and disease-free survival. In cases classified as FIGO stage Ib (n = 35), after adjusting for patient age at diagnosis and stratifying by histological grade, MUC1 and/or MUC 16 overexpression was an independent prognostic factor for overall survival (hazard ratio [HR] = 6.16, 95% confidence interval [CI] = 1.01–118.5, P

Multifunctional macrophage inhibitory cytokine-1, MIC-1, is a member of the transforming growth factor-beta (TGF-beta) superfamily that plays key roles in the prenatal development and regulation of the cellular responses to stress signals and inflammation and tissue repair after acute injuries in adult life. The stringent control of the MIC-1 expression, secretion, and functions involves complex regulatory mechanisms and the interplay of other growth factor signaling networks that control the cell behavior. The deregulation of MIC-1 expression and signaling pathways has been associated with diverse human diseases and cancer progression. The MIC-1 expression levels substantially increase in cancer cells, serum, and/or cerebrospinal fluid during the progression of diverse human aggressive cancers, such as intracranial brain tumors, melanoma, and lung, gastrointestinal, pancreatic, colorectal, prostate, and breast epithelial cancers. Of clinical interest, an enhanced MIC-1 expression has been positively correlated with poor prognosis and patient survival. Secreted MIC-1 cytokine, like the TGF-beta prototypic member of the superfamily, may provide pleiotropic roles in the early and late stages of carcinogenesis. In particular, MIC-1 may contribute to the proliferation, migration, invasion, metastases, and treatment resistance of cancer cells as well as tumor-induced anorexia and weight loss in the late stages of cancer. Thus, secreted MIC-1 cytokine constitutes a new potential biomarker and therapeutic target of great clinical interest for the development of novel diagnostic and prognostic methods and/or cancer treatment against numerous metastatic, recurrent, and lethal cancers.

Mucins are the most abundant high molecular weight glycoproteins in mucus. Their nature and glycosylation content dictates the biochemical and biophysical properties of viscoelastic secretions, pointing out an important role in diverse biological functions, such as differentiation, cell adhesions, immune responses, and cell signaling. Mucins are expressed in tubular organs by specialized epithelial cells in the body. Their aberrant expression is well documented in a variety of inflammatory or malignant diseases. From a prognosis point of view, their expression and alterations in glycosylation are associated with the development and progression of malignant diseases. Therefore, mucins can be used as valuable markers to distinguish between normal and disease conditions. Indeed, this alteration in glycosylation patterns generates several epitopes in the oligosaccharide side chains that can be used as diagnostic and/or prognostic markers. Furthermore, these characteristic tumor-associated epitopes are extensively used as appropriate immunotargets of malignant epithelial cells. Therefore, in an effort to detect and treat cancer at the earliest stage possible, mucins are analyzed as potential markers of disease for diagnosis, progression, and for therapeutic purposes. In this review, we focused on the current status of the distribution of mucins in normal and pathologic conditions and their clinical use both in cancer diagnosis and therapeutics treatments.

Recent advances in skin-resident adult stem/progenitor cell research have revealed that these immature and regenerative cells with a high longevity provide critical functions in maintaining skin homeostasis and repair after severe injuries along the lifespan of individuals. The establishment of the functional properties of distinct adult stem/progenitor cells found in skin epidermis and hair follicles and extrinsic signals from their niches, which are deregulated during their aging and malignant transformation, has significantly improved our understanding on the etiopathogenesis of diverse human skin disorders and cancers. Particularly, enhanced ultraviolet radiation exposure, inflammation and oxidative stress and telomere attrition during chronological aging may induce severe DNA damages and genomic instability in the skin-resident stem/progenitor cells and their progenies. These molecular events may result in the alterations in key signalling components controlling their self-renewal and/or regenerative capacities as well as the activation of tumour suppressor gene products that trigger their growth arrest and senescence or apoptotic death. The progressive decline in the regenerative functions and/or number of skin-resident adult stem/progenitor cells may cause diverse skin diseases with advancing age. Moreover, the photoaging, telomerase re-activation and occurrence of different oncogenic events in skin-resident adult stem/progenitor cells may also culminate in their malignant transformation into cancer stem/progenitor cells and skin cancer initiation and progression. Therefore, the anti-inflammatory and anti-oxidant treatments and stem cell-replacement and gene therapies as well as the molecular targeting of their malignant counterpart, skin cancer-initiating cells offer great promise to treat diverse skin disorders and cancers.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with etiological association with cigarette smoking. Nicotine, an important component of cigarettes, exists at high concentrations in the bloodstream of smokers. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. Here, we investigated the potential molecular basis of nicotine's role in PDA through studying its effect on OPN. Nicotine significantly (p < 0.02) increased OPN mRNA and protein secretion in PDA cells through activation of the OPN gene promoter. The OPN mRNA induction was inhibited by the nicotinic acetylcholine receptor antagonist, mechamylamine. Further, the tyrosine kinase inhibitor genistein inhibited the nicotine-mediated induction of OPN, suggesting that mitogen activated protein kinase signaling mechanism is involved. Nicotine activated the phosphorylation of ERK1/2, but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the nicotine-induced OPN synthesis. Rats exposed to cigarette smoke showed a dose-dependent increase in pancreatic OPN that paralleled the rise of pancreatic and plasma nicotine levels. Analysis of cancer tissue from invasive PDA patients, the majority of whom were smokers, showed the presence of significant amounts of OPN in the malignant ducts and the surrounding pancreatic acini. Our data suggest that nicotine may contribute to PDA pathogenesis through upregulation of OPN. They provide the first insight into a nicotine-initiated signal transduction pathway that regulates OPN as a possible tumorigenic mechanism in PDA.

Recent advances in basic and clinical oncology have led to the identification and characterization of several deregulated gene products involved in cancer progression to invasive, metastatic, and recurrent disease stages. Particularly, an emerging body of experimental evidence has revealed that an accumulation of genetic and/or epigenetic alterations can lead to the malignant transformation of tissue-resident adult stem/progenitor cells into leukemic or tumorigenic cancer stem/progenitor cells. These immature cancer cells endowed with a self-renewal capacity and aberrant differentiation potential that can drive leukemia and tumor development and metastases being responsible for treatment resistance and disease relapse. Therefore, the molecular targeting of deregulated signaling elements that may contribute to the uncontrolled growth, survival, invasion, and resistance of these cancer-initiating cells to current clinical cancer therapies is of great therapeutic interest. Potential anti-cancer drug targets include telomerase, anti-apoptotic factors, DNA repair, and detoxifying enzymes and ATP-binding cassette multidrug transporters as well as distinct oncogenic cascades, including hedgehog, EGFR, Wnt/β-catenin, Notch, and/or polycomb group (PcG) protein chromatin-silencing pathways. The combined targeting of these deregulated gene products should reverse multidrug resistance and eliminate the cancer-initiating cells and their differentiated progenies. Hence, these novel targeting approaches could be used to improve the efficacy of current therapeutic treatments against the highly aggressive, metastatic, recurrent and lethal cancers. Drug Dev Res 69:415–430, 2008. © 2008 Wiley-Liss, Inc.

Overcoming intrinsic and acquired resistance of cancer stem/progenitor cells to current clinical treatments represents a major challenge in treating and curing the most aggressive and metastatic cancers. This review summarizes recent advances in our understanding of the cellular origin and molecular mechanisms at the basis of cancer initiation and progression as well as the heterogeneity of cancers arising from the malignant transformation of adult stem/progenitor cells. We describe the critical functions provided by several growth factor cascades, including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor (SCF) receptor (KIT), hedgehog and Wnt/β -catenin signalling pathways that are frequently activated in cancer progenitor cells and are involved in their sustained growth, survival, invasion and drug resistance. Of therapeutic interest, we also discuss recent progress in the development of new drug combinations to treat the highly aggressive and metastatic cancers including refractory/relapsed leukaemias, melanoma and head and neck, brain, lung, breast, ovary, prostate, pancreas and gastrointestinal cancers which remain incurable in the clinics. The emphasis is on new therapeutic strategies consisting of molecular targeting of distinct oncogenic signalling elements activated in the cancer progenitor cells and their local microenvironment during cancer progression. These new targeted therapies should improve the efficacy of current therapeutic treatments against aggressive cancers, and thereby preventing disease relapse and enhancing patient survival.

BACKGROUND The expression of prostate-derived factor (PDF) is significantly elevated in human prostate tumors. We investigate the functional role and signaling of PDF in androgen receptor (AR)-positive human prostate cancer cells. METHODS Transient or stable expression of PDF by cDNA transfection, antisense-mediated gene silencing, media conditioned by PDF-elevated cells, and antibody (Ab) neutralization were employed. RESULTS Elevated endogenous and exogenous expression of PDF and treatment of PDF-enriched media were associated with increased proliferation and clonogenic growth of the cells. On the contrary, knockdown of PDF or addition of PDF neutralizing Ab resulted in diminished proliferation and reduced anchorage-independent growth. Further, ERK1/2 and p90RSK, but not Smad2/3, were activated in PDF-elevated cells as well as in cells treated with PDF-enriched media, while inhibition of ERK1/2 decreased the growth of those cells. CONCLUSION PDF promotes AR-positive prostate tumor progression through upregulating cell proliferation via ERK1/2 signal pathway. Prostate 67: 557–571, 2007. © 2007 Wiley-Liss, Inc.

BACKGROUND Mucins are considered important markers for early diagnosis and targeted therapy due to their aberrant and unique expression pattern during malignant progression of carcinomas. Recent findings have provided substantial evidence for the involvement of transmembrane mucins, MUC1 and MUC4, in altered cell signaling, tumor growth, and metastasis. METHODS Immunohistochemical analyses were performed on prostate tumor tissues for expression profiling of the two transmembrane mucins, MUC1 and MUC4. In cancer cell lines, the expression was studied by RT-PCR and immunoblot analyses. Cells were treated with DNA-methylase and histone-deacetylase inhibitors to examine the implication of epigenetic mechanism(s) in MUC4 regulation. RESULTS The expression of MUC4 was significantly downregulated in prostate cancer tissues (n = 38, P = 0.0026) compared to normal/benign prostatic hyperplastic regions. A faint to moderate staining was observed in 26.3% cases of cancer, while 84.2% cases of adjacent normal were positive for MUC4 with moderate to strong staining in most cases. Similar observations were made in immortalized normal prostate epithelial and cancer cell lines. MUC1 also showed a reduced expression in prostate tumor tissues; however, its expression was comparable in all normal prostate epithelial and cancer cell lines. Interestingly, we also found that epigenetic mechanism(s) might be implicated in MUC4 gene silencing. CONCLUSIONS Our data suggest that MUC4 downregulation may be of significance for diagnostic applications in prostate cancer. © 2005 Wiley-Liss, Inc.

Although the blockade of the hedgehog cascade by using cyclopamine has been reported to inhibit the growth of some cancer cell types, few studies on the mechanism by which this drug alone or in combination with other cytotoxic agents induces its cytotoxic effect have been reported. In our study, we evaluate, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective SMO inhibitor, cyclopamine and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib on metastatic prostate cancer (PC) cells. The results revealed that cyclopamine, alone or at a lower concentration in combination with gefitinib, inhibited the growth of sonic hedgehog- (SHH), epidermal growth factor- (EGF) and serum-stimulated androgen-sensitive LNCaP-C33 and LNCaP-LN3 and androgen-independent LNCaP-C81, DU145 and PC3 cells. The antiproliferative effect of cyclopamine and gefitinib, alone or in combination, was mediated via a blockade of the PC3 cells in the G1 phase of the cell cycle. Importantly, the combined cyclopamine and gefitinib also caused a higher rate of apoptotic death of PC cells compared to single agents. The cytotoxic effect induced by these drugs in PC3 cells appears to be mediated at least, in part, via the mitochondrial pathway through the depolarization of the mitochondrial membrane and the release of cytochrome c and reactive oxygen species into the cytosol. This was also accompanied by the activation of caspase cascades, PARP cleavage and DNA fragmentation. Additionally, the combined cyclopamine and gefitinib were more effective at suppressing the invasiveness of PC3 cells through matrigel in vitro as the drugs alone. These findings indicate that the simultaneous blockade of SHH-GLI-1 and EGF-EGFR signaling, which results in the growth arrest and massive rate of apoptotic cell death, represents a promising strategy for a more effective treatment of metastatic PC forms.

Molecular genetic mechanisms involved in the progression of prostate cancer are not well understood due to extensive tumor heterogeneity and lack of suitable models. New methods such as fluorescence in-situ hybridization (FISH), comparative genomic hybridization (CGH) and microsatellite analysis have documented losses or gains on various chromosomes. Altered chromosomal regions have been associated with the activation of oncogenes and the inactivation of tumor suppressor genes or defects in mismatch repair (MMR) genes. It is suggested that increased genomic instability is associated with decreased androgen-responsive and progressive behavior of human prostate tumors, but it remains unclear whether this genomic instability is causing the progression of cancer or is the consequence of cancer. Extended studies on hereditary prostate cancer have identified 7 prostate cancer susceptibility loci on several chromosomes, but no specific gene has been confined for a large proportion of susceptibility. In this review we summarize the ongoing molecular genetic events associated with the sporadic and hereditary prostate cancer development and progression.

The human MUC4 gene encodes a large membrane-associated mucin, characterized by a mucin tandem repeat domain and a growth factor-like transmembrane domain. In addition to the originally published sequence (sv0-MUC4), several MUC4 cDNA sequences (called sv1-MUC4 to sv21-MUC4, MUC4/X, MUC4/Y) from various tissues and cell lines have been recently described. They differ from sv0-MUC4 by deletions and/or insertions located in the 3' region or, for two of them, by deletion of the central repetitive domain. To establish the nature of the mechanisms responsible for the diversity of MUC4 transcripts, the genomic structure of the 3' region of the human MUC4 gene was determined. Our results show that it spans approximately 30.8 kb of genomic DNA and is composed of 24 exons, including one alternative exon which was exclusively reported for sv1-MUC4. Moreover, we have shown that the different MUC4 transcripts are generated by several mechanisms, including the alternative use of cassette exons, exon skipping or use of cryptic splice donor/acceptor sites.

BACKGROUND The acquisition of an androgen-independent phenotype is the most serious issue of prostate cancer treatment. Although several experimental cell models have been reported for studying androgen independence, they have limited applications related to hormone-refractory prostate cancer. To investigate the molecular mechanism of androgen-independent growth of prostate cancer, we established a useful LNCaP cell model that resembles the clinical scenario of hormone-refractory prostate cancer. METHODS Androgen-sensitive LNCaP parental cells were continuously maintained in a regular cell-culture medium, that is, phenol red–positive RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% glutamine. Upon passage, the androgen responsiveness of those cells decreased, to a level lower than that of parental cells. We examined the growth properties and androgen responsiveness of these different LNCaP cells in vitro and in vivo. Cytogenetic characteristics and expression of androgen receptors (ARs) and prostate-specific antigen (PSA) were determined. RESULTS Upon continuous passage, the biological behavior of parental C-33 cells (passage number less than 33) was altered. C-81 cells (passage number higher than 81) clearly exhibited more aggressive growth and lower androgen responsiveness than C-33 and C-51 cells (passage number between 35 and 80) in vitro and in vivo. Nevertheless, all these cells expressed a similar level of functional AR protein as well as a similar genetic profile. Moreover, in a steroid-reduced culture condition, C-81 cells secreted a higher level of PSA than C-33 cells. CONCLUSIONS Our LNCaP cell model closely recapitulates the progression of human prostate cancer from the androgen-responsive to the hormone-refractory state under the androgen nondeprived condition. This cell model may provide the opportunity to understand the molecular mechanisms associated with the acquisition of androgen independence during human prostate cancer progression. Prostate 50: 222–235, 2002. © 2002 Wiley-Liss, Inc.

One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.

The MUC4 mucin gene encodes a putative membrane-anchored mucin with predicted size of 930 kDa, for its 26.5-kb allele. It is composed of two regions, the 850-kDa mucin-type subunit MUC4alpha and the 80-kDa membrane-associated subunit MUC4beta. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1-MUC4 to sv8-MUC4, the full length cDNA being called sv0-MUC4) provided seven distinct variants, five secreted forms and two membrane-associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane-bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.

Pancreatic cancer (PC) is a common disease that is seldom cured. Current approaches to the treatment of PC are not effective because the non-specific nature of both chemotherapy and external beam radiation results in toxicity to normal tissue. Monoclonal antibodies (MAbs) can be used as selective carriers for delivering radionuclides, toxins, or cytotoxic drugs to malignant cell populations. Therefore, MAb-technology has led to a significant amount of research in targeted therapy. Targeted therapy would generally allow the concentration of cytotoxic agents in tumors and would markedly lessen the toxicity to normal tissues, which limits the dosage and effectiveness of systemically administered drugs. A variety of MAbs are being pre-clinically evaluated for the diagnosis and treatment of PC. Novel recombinant antibody constructs hold a promising future in both the diagnosis and treatment of cancer. By genetic-engineering methods, several high affinity antibody fragments with optimum tumor targeting properties, such as higher functional affinity (divalent and multivalent scFvs) and blood residence time (good tumor localization with high radiolocalization index), have been generated. Animal models have permitted the in vivo assessment of these antibody-based reagents, therapeutic/diagnostic radionuclide, radiolabeling conditions, and efficacy of administration regimes. For PC, immunoscintigraphy using MAbs has taken new strides. The use of MAbs and their fragments for radioimmunoguided surgery and therapy of PC has shown encouraging results at preclinical levels and warrants further attention.

CD44 is an integral cell-surface glycoprotein. Overexpression of the CD44 standard (CD44st) and its variants (CD44v) has been implicated in transformation and progression of many cancer types. Here, we investigated expression of CD44st, CD44v3-7, CD44v7/8, and v10 in five human pancreatic tumor cell lines and normal human pancreatic duct cells transfected with the SV40 large T antigen. CD44st and its variant proteins were quantified using immunocytochemistry and flow cytometry. CD44v7 was expressed at low levels, whereas CD44st, CD44v3, CD44 v4, CD44v, and CD44v6 were expressed at moderate levels in all pancreatic tumor cell lines. In contrast, CD44v7/8 and CD44v10 were expressed at very low levels in two out of the five pancreatic tumor cell lines. Overall, staining of CD44st and CD44 variants was significantly weaker compared to another surface molecule, ICAM-1, reported to be overexpressed in pancreatic cancer cells. Furthermore, the SV40 large T transfected duct cells showed only a weak staining for CD44st, CD44v5, and CD44v6. To determine a possible mechanism for the regulation of surface expression of CD44st, v5 and v6, we incubated Panc-1 cells with bFGF, TGF-beta1, EGF, TNFalpha, and IFNgamma. Only IFNgamma affected the CD44 expression by down-regulation of CD44v6. The constitutive expression of CD44 variants seems to be associated with the malignant state of invasive carcinoma.

MUC4 is a membrane-bound mucin and is considered as the human homologue of the rat sialomucin complex (SMC). The deduced structural organization of the wild type-MUC4 cDNA (WT-MUC4) sequence revealed two subunits: a large amino mucin type subunit (MUC4alpha) and a transmembrane subunit (MUC4beta). MUC4beta is a membrane-bound growth factor like subunit and contains three EGF-like domains. The MUC4 gene is expressed in several normal tissues like trachea, lung, and testis. It is not expressed in a normal human pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors and tumor cell lines. Recently, we have demonstrated the presence of alternative splice events in the 3'-end of the MUC4 cDNA that generated new putative variants (sv1-sv10) in normal human testis and in a pancreatic tumor cell line (HPAF). In search of MUC4 variant(s) that are specific to pancreatic adenocarcinoma, we investigated the splicing phenomena in the MUC4 cDNA sequence by using a large panel of pancreatic tumor cell lines. We have identified ten alternative splice events located downstream to the central large tandem repeat domain. These splice events generated 12 variant species (sv4, sv9, sv10-18, and sv21) of MUC4 cDNAs. The deduced amino acid sequence of these variant MUC4 cDNAs revealed two distinct types: a family of secreted and a membrane-associated variant form. Among the members of MUC4 secreted variant family, three (sv4, sv12, and sv13) of ten showed a short 144 residue COOH-terminus compared to 1154 residues in WT-MUC4. The variants with this short COOH-terminus (144 residues) was found in 37% (4/11) of the tumor lines. The putative membrane-bound variant sv10 was detected in 37% (4/11) pancreatic tumor cell lines but not in any normal human tissues. In conclusion, we have identified novel splice variant(s) of MUC4 in pancreatic adenocarcinoma.

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv)2 exhibited a relative KA (3.34 × 107 M−1) similar to that of the native IgG (1.14 × 108 M−1) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv)2, with a T1/2 less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv)2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv)2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications. Int. J. Cancer 81:911–917, 1999. © 1999 Wiley-Liss, Inc.

Pancreatic cancer is a therapeutic challenge for surgical and medical oncology. Development of specific molecular tracers for the diagnosis and treatment of this lethal cancer has been one of our major goals. Monoclonal antibodies (MAbs) have been successfully used as selective carriers for delivering radionuclides, toxins or cytotoxic drugs to malignant cell populations; therefore, monoclonal antibody technology has led to a significant amount of research into optimizing targeted therapy. This targeted therapy results in the selective concentration of cytotoxic agents or radionuclides in tumors and should lessen the toxicity to normal tissues, which would normally limit the dosage and effectiveness of systemically administered drugs. The MAb CC49 reacts with a unique disaccharide, Sialyl-Tn, present on tumor-associated mucin (TAG-72) expressed by a majority of human adenocarcinomas. The unique Sialyl-Tn epitope has provided a potential target for immunotherapy of cancer. A single chain Fv (scFv) recombinant protein from CC49 MAb was prepared by engineering the DNA fragments for coding heavy-chain and light-chain variable regions with an appropriate oligonucleotide linker. scFv molecules, when compared to intact MAbs and the more conventional enzymatically derived F(ab')2 and Fab' fragments, offer several advantages as carriers for the selective delivery of radionuclides to tumors. The divalent antibody fragments (sc(Fv)2 or (scFv)2) display an affinity constant similar to that of the intact CC49 IgG and are stable with storage, and after radiolabeling. In preclinical studies, both the covalent and the non-covalent dimeric scFvs exhibit excellent tumor targeting properties with characteristics similar to those of the monomer, e.g., the rapid blood clearance, low kidney uptake and small size suitable for rapid penetration through tumor tissue. Increased tumor targeting of the dimers are probably due to their increased functional affinity attributable to valency, coupled with their higher molecular weight and fewer interactions with normal organs. These properties make these constructs superior to monovalent CC49 scFv. The relatively high tumor uptake, the in vitro and in vivo targeting specificity, and the stability in storage demonstrated by the dimeric CC49 sc(Fv)2 makes it a promising delivery vehicle for therapeutic applications in pancreatic cancer.

Loss of heterozygosity at specific chromosomal locations has been taken as evidence of a tumor suppressor gene located in that area. We performed a genomic allelotyping study on 46 childhood brain tumors of different histopathological types in order to identify and confirm common areas of deletion in different tumor types. Two hundred microsatellite DNA probes equally distributed over the 22 autosomes were applied, covering the genome in steps of approximately 25 cM. Our results confirm frequent loss of heterozygosity of chromosome arms 9q, 10q, 11p, 11q, 16q, and 22q in high-grade gliomas, medulloblastomas, and ependymomas. In addition, we found a new region of loss on chromosome segment 2p21-23 affected predominantly in high-grade gliomas and medulloblastomas.

Antibodies by virtue of their unmatched specificity and diverse modes of action are the central components of the immune system to detect and combat pathogenic and nonpathogenic diseases. From the era of polyclonal antibodies to fully humanised monoclonal antibodies, a lot has been understood regarding their causal generation, molecular buildup, varied functioning and clearance from the biological system. This information has led to their widespread use in various arenas of pathogenesis including parasitic, bacterial and viral infections, heart diseases, autoimmune diseases and cancer. Since cancer derives from self, it is both difficult to diagnose as well as treat. With over 20 antibody-based drugs now in use and over 100 in clinical trials for cancer, antibodies form the basis of several diagnostic as well as therapeutic modalities for some lethal malignancies. Further, antibody engineering to improve their characteristics, such as pharmacokinetics, has made them a major attraction for the biopharmaceutical industry. Key Concepts: The efficacy of murine monoclonal antibodies is limited by the inefficient action of the murine-derived constant region and human anti-mouse antibody response, resulting in their rapid clearance from the blood. The success of an antibody-based biopharmaceutical depends upon target antigen (location, distribution and density), targeted site (solid or haematological malignancy, tumour microenvironment) and targeting antibody (specificity, affinity, avidity, mode of action, origin and isotype). Antitumour antibodies mediate their effector functions by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis. Antigenic heterogeneity, lack of tumour specificity, immunogenicity and nonuniform distribution of antibodies to the targeted site are major hurdles in successful use of antibody-based therapeutics for solid tumours. Antibodies against molecules inhibiting effector functions of helper and cytotoxic T cells improve the efficacy of an antitumour immune response. Antibodies directed against co-stimulatory receptors act as surrogate ligands and boost weak, ineffectual and endogenous antitumour immune responses. Keywords: tumour targeting; antibodies; imaging; monoclonal; immunoscintigraphy

Complex signaling cross-talks between different growth factor cascades orchestrate the primary brain cancer development. Among the frequent deregulated oncogenic pathways, the ligand-activated wild-type epidermal growth factor receptor (EGFR), constitutively activated EGFRvIII mutant and sonic hedgehog pathways have attracted much attention because of their pivotal roles in pediatric medulloblastomas and adult glioblastoma multiformes (GBM) brain tumors. The enhanced expression levels and activation of EGFR, EGFRvIII mutant and hedgehog signaling elements can provide key roles for the sustained growth, migration and local invasion of brain tumor-initiating cells (BTICs) and their progenies, resistance to current therapies and disease relapse. These tumorigenic cascades also can cooperate with Wnt/β-catenin, Notch, platelet-derived growth factor (PDGF)/PDGF receptors (PDGFRs), hepatocyte growth factor (HGF)/c-Met receptor and vascular endothelial growth factor (VEGF)/VEGF receptors (VEGFRs) for the acquisition of a more malignant behavior and survival advantages by brain tumor cells during disease progression. Therefore, the simultaneous targeting of these oncogenic signaling components including wild-type EGFR, EGFRvIII mutant and hedgehog pathways may constitute a potential therapeutic approach of great clinical interest to eradicate BTICs and improve the efficacy of current clinical treatments by radiation and/or chemotherapy against aggressive and recurrent medulloblastomas and GBMs.

The transcription, which is the RNA (ribonucleic acid) biosynthesis, constitutes a tightly regulated multistep process involving the spatial and temporal recruitment of numerous protein complexes. The three main steps of the process are transcription initiation with the formation of the transcription preinitiation complex (PIC), elongation and termination. All these steps have in common the enzymatic activity of the protein complex known as the RNA polymerase II (RNAPII), which is an enzyme giving rise to the promoter-specific protein complex called RNAPII holoenzyme. The composition of RNAPII holoenzyme (dependent on the extraction/purification process and the origin of the material) varies from laboratory to laboratory, but always includes RNAPII, general transcription factors (GTFs), protein mediators such as the suppressor of RNA polymerase B mutations (SRBs), histone acethyl transferase (HAT) and chromatin remodelling factors. This article focuses on the structure of RNAPII and the protein complex leading to the formation of RNAPII holoenzyme during the initiation step. The role of the main proteins allowing specific promoter recognition is detailed. Key concepts: The transcription is a regulated multistep process (initiation, elongation and termination) leading to RNA synthesis. RNA polymerase synthesizes RNA in a DNA-dependent manner. Six RNA polymerases are known in eukaryote: RNAPI (synthesizing rRNAs), RNAPII (synthesizing mRNAs and snRNAs), RNAPIII (synthesizing tRNAs, 5S rRNA and some snRNAs), spRNAP-IV (synthesizing mRNAs) and RNAP IV (synthesizing siRNA). Transcription requires cis-acting promoter elements (core and regulatory elements) and trans-acting factors. The core elements bind the transcription preinitiation complex (PIC) that include the GTFs (general transcription factor) and RNAPII. Two main forms of RNAPII holoenzyme are identified, the SRB/MED-containing and the PAF complex-containing holoenzyme. Keywords: transcription; initiation; RNA polymerase II; general transcription factor; core promoter; holoenzyme