Emerging Insights into Wnt/ß-catenin Signaling in Head and Neck Cancer.

Head and neck cancer presents primarily as head and neck squamous cell carcinoma (HNSCC), a debilitating malignancy fraught with high morbidity, poor survival rates, and limited treatment options. Mounting evidence indicates that the Wnt/ß-catenin signaling pathway plays important roles in the pathobiology of HNSCC. Wnt/ß-catenin signaling affects multiple cellular processes that endow cancer cells with the ability to maintain and expand immature stem-like phenotypes, proliferate, extend survival, and acquire aggressive characteristics by adopting mesenchymal traits. A central component of canonical Wnt signaling is ß-catenin, which balances its role as a structural component of E-cadherin junctions with its function as a transcriptional coactivator of numerous target genes. Recent genomic characterization of head and neck cancer revealed that while ß-catenin is not frequently mutated in HNSCC, its activity is unchecked by more common mutations in genes encoding upstream regulators of ß-catenin, NOTCH1, FAT1, and AJUBA. Wnt/ß-catenin signaling affects a wide range epigenetic and transcriptional activities, mediated by the interaction of ß-catenin with different transcription factors and transcriptional coactivators and corepressors. Furthermore, Wnt/ß-catenin functions in a network with many signaling and metabolic pathways that modulate its activity. In addition to its effects on tumor epithelia, ß-catenin activity regulates the tumor microenvironment by regulating extracellular matrix remodeling, fibrotic processes, and immune response. These multifunctional oncogenic effects of ß-catenin make it an attractive bona fide target for HNSCC therapy.

A comprehensive analysis of gene expression profiles in a yeast N-glycosylation mutant.

Although protein N-glycosylation is critical to many cell functions, its downstream targets remain largely unknown. In all eukaryotes, N-glycosylation utilizes the lipid-linked oligosaccharide (LLO) precursor, whose synthesis is initiated by the ALG7 gene. To elucidate the key signaling and metabolic events affected by N-glycosylation, we performed genomewide expression profiling of yeast cells carrying a hypomorphic allele of ALG7. DNA microarrays showed that of more than 97% of known or predicted yeast genes, 29 displayed increased expression while 23 were repressed in alg7 mutants. Changes in transcript abundance were observed for a and alpha mating-type genes, for genes functioning in several mitogen-activated protein kinase (MAPK) cascades, as well as in phosphate, amino acid, carbohydrate, mitochondrial and ATP metabolism. Therefore, DNA microarrays have revealed direct and indirect targets, including internal feedback loops, through which N-glycosylation affects signaling and metabolic activities and is functionally linked with cellular regulatory circuitry.

Loss of alpha3beta1 integrin function results in an altered differentiation program in the mouse submandibular gland.

Mammalian submandibular gland (SMG) development leads to the establishment of highly organized secretory acinar and nonsecretory ductal epithelial cells. The ability of maturing salivary epithelial cells to attain their differentiated state has been shown to depend, in part, on interactions between extracellular matrix (ECM) proteins and their integrin receptors. In a search for key regulators of salivary cell lineage, we have studied alpha3beta1 integrin, a receptor for the basement membrane protein laminin, by characterizing embryonic day 18 (E18) SMGs isolated from mice carrying a targeted mutation in the alpha3 integrin gene. Transmission electron microscopy studies showed that the mutant SMGs exhibited an aberrant differentiation phenotype with defects in the apical-basal polarity axis and in the basement membrane. Based on immunohistochemistry and Western blot analyses, the alpha3beta1-deficient SMGs had altered expression and/or localization of several ECM and adhesive molecules, including laminin beta1, fibronectin, alpha5 integrin, and E-cadherin. These changes correlated with alterations in the activation state of Ras-extracellular signal-regulated kinase (ERK), as well as the expression and/or localization of Cdc42 and RhoA, two Rho GTPases that regulate the organization of the actin cytoskeleton. We conclude that alpha3beta1 is required for normal salivary cell differentiation and that its absence affects multiple components of adhesive complexes and their associated signalling pathways.

Differential expression of proliferative, cytoskeletal, and adhesive proteins during postnatal development of the hamster submandibular gland.

Although the submandibular gland (SMG) plays important exocrine and endocrine roles, little is known about the molecular details underlying its development. Previously, we reported that in the postnatally developing hamster SMG, GPT, the protein product of the first N-glycosylation gene, ALG7, was an in vivo marker for salivary cell proliferation. Here we investigated the proliferative, cytoskeletal, and adhesive changes during SMG postnatal development. The cellular localization and abundance of GPT, filamentous actin, and beta1 integrin receptor were examined using confocal microscopy and immunoblotting. In neonatal glands, high GPT levels marked extensive cell proliferation throughout the tissue. The apical regions of immature salivary cells displayed intense actin staining, while most of the beta1 integrin was diffusely distributed throughout the tissue. As development proceeded, discrete regions of the gland expressed attenuated levels of GPT, an increased organization of actin to the cell cortex, and beta1 integrin to the basal lamina. In the adult SMG, differentiated salivary cells displayed low levels of GPT and actin. While the abundance of beta1 integrin remained unchanged throughout development, in the adult, it was found exclusively in regions where cells contact the basal lamina. These data indicate that SMG development entails regionalized cell proliferation and polarization, and that these processes are temporally and spatially coordinated with the establishment of stable cell-substratum interactions.

ALG gene expression and cell cycle progression.

The evolutionarily conserved ALG genes function in the dolichol pathway in the synthesis of the lipid-linked oligosaccharide precursor for protein N-glycosylation. Increasing evidence suggests a role for these genes in the cell cycle. In Saccharomyces cerevisiae, coordinate regulation of the ALG genes makes up the primary genomic response to growth stimulation; several features of the ALG genes' expression resemble mammalian early growth response genes. However, only the first gene in the pathway, ALG7, is downregulated in response to an antimitogenic signal that leads to cell cycle arrest and differentiation, suggesting that selective inhibition of the first gene may be sufficient to regulate the dolichol pathway for the withdrawal from the cell cycle. The availability of mutants in the early essential ALG genes has established functional relationships between these genes' expression and G1/S transition, budding, progression through G2 and withdrawal from the cell cycle. Analysis of the regulation of ALG7 has provided insights into how this gene's expression is controlled at the molecular level. Recent studies have also begun to reveal how ALG7 expression is linked to cell cycle arrest in response to antimitogenic cues and have identified G1 cyclins as some of its downstream targets. Since the functions of the ALG genes appear to be as conserved among eukaryotes as the cell cycle machinery, it is likely that these genes play a similar role in mammalian cell proliferation and differentiation.

Protein N-glycosylation: molecular genetics and functional significance.

Protein N-glycosylation is a metabolic process that has been highly conserved in evolution. In all eukaryotes, N-glycosylation is obligatory for viability. It functions by modifying appropriate asparagine residues of proteins with oligosaccharide structures, thus influencing their properties and bioactivities. N-glycoprotein biosynthesis involves a multitude of enzymes, glycosyltransferases, and glycosidases, encoded by distinct genes. The majority of these enzymes are transmembrane proteins that function in the endoplasmic reticulum and Golgi apparatus in an ordered and well-orchestrated manner. The complexity of N-glycosylation is augmented by the fact that different asparagine residues within the same polypeptide may be modified with different oligosaccharide structures, and various proteins are distinguished from one another by the characteristics of their carbohydrate moieties. Furthermore, biological consequences of derivatization of proteins with N-glycans range from subtle to significant. In the past, all these features of N-glycosylation have posed a formidable challenge to an elucidation of the physiological role for this modification. Recent advances in molecular genetics, combined with the availability of diverse in vivo experimental systems ranging from yeast to transgenic mice, have expedited the identification, isolation, and characterization of N-glycosylation genes. As a result, rather unexpected information regarding relationships between N-glycosylation and other cellular functions--including secretion, cytoskeletal organization, proliferation, and apoptosis--has emerged. Concurrently, increased understanding of molecular details of N-glycosylation has facilitated the alignment between N-glycosylation deficiencies and human diseases, and has highlighted the possibility of using N-glycan expression on cells as potential determinants of disease and its progression. Recent studies suggest correlations between N-glycosylation capacities of cells and drug sensitivities, as well as susceptibility to infection. Therefore, knowledge of the regulatory features of N-glycosylation may prove useful in the design of novel therapeutics. While facing the demanding task of defining properties, functions, and regulation of the numerous, as yet uncharacterized, N-glycosylation genes, glycobiologists of the 21st century offer exciting possibilities for new approaches to disease diagnosis, prevention, and cure.

Molecular dissection of the genetic targets of ALG7 in the serpentine receptor-mediated signal transduction pathway in yeast.

These initial studies show that deregulated expression of ALG7 affects diverse cellular functions crucial to development, including proliferation, differentiation, and morphogenesis. Furthermore, the data suggest multiple genetic targets for ALG7 and provide the basis for future dissection of these developmentally relevant pathways.

Confocal imaging of gene expression during hamster submandibular gland biogenesis.

The data presented here provide evidence that the abundance of the ALG7 protein product, GPT, correlates with high proliferative activity during the postnatal development of the hamster SMG development, and that it becomes downregulated with differentiation. Based on our previous studies with yeast, changes in the level of ALG7 expression may be necessary for the events directing salivary cell polarization, migration, differentiation, and apoptosis at distinct developmental stages.

Characterization of multiple transcripts of the hamster dolichol-P-dependent N-acetylglucosamine-1-P transferase suggests functionally complex expression.

The evolutionarily conserved dolichol-P-dependent N-acetylglucosamine-1-P transferase gene, ALG7, functions by initiating the dolichol pathway of protein N-glycosylation. In yeast, ALG7 has a complex expression pattern and plays a critical role in diverse cellular functions, including proliferation and morphological response. In Chinese hamster ovary cells (CHO), ALG7 gives rise to three mRNAs of 1.5, 1.9 and 2.2 kb. We report results of RNA blotting assays, ribonuclease protection, PCR-amplification and sequencing of the CHO ALG7 transcripts 5' and 3' ends which suggest that the 1.5 and 1.9 kb transcripts are produced as a consequence of initiation at 2 distinct start sites, 350-379 bp apart. The transcriptional start site for the 1.5 kb mRNA is positioned between the first two in frame ATGs, while that of the 1.9 kb species is located upstream of these two in-frame ATGs. In order to test the translational competence of the 1.5 and 1.9 kb mRNAs, we constructed DNA templates specifying these transcripts and used them for in vitro transcription/translation. Our data show that the 1.9 kb mRNA served in the synthesis of 36 and 24 kDa species, as well as a low-abundance 32 kDa protein. The 1.5 kb transcript gave rise to a translation product of 32 kDa. The latter is synthesized in CHO cells and hamster submandibular glands. These results suggest the possibility that the 1.5 and 1.9 kb transcripts give rise to related protein isoforms with different lengths of their NH2-terminal regions.

Deregulation of the first N-glycosylation gene, ALG7, perturbs the expression of G1 cyclins and cell cycle arrest in Saccharomyces cerevisiae.

The evolutionarily conserved ALG7 gene encodes the dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT) and functions by initiating the dolichol pathway of protein N-glycosylation. In Saccharomyces cerevisiae, ALG7 has been shown to play a role in cell proliferation. The yeast alpha-factor-induced cell cycle arrest in G1 occurs, in part, by downregulation of CLN1 and CLN2. The function of ALG7 in G1 arrest was examined in alg7 mutants containing diminished GPT activity. In wild type, CLN1 and CLN2 mRNAs were rapidly downregulated, while in alg7 mutants, these transcripts were only transiently repressed before becoming greatly augmented. Analyses of DNA contents and budding indices showed that alg7 mutants resumed cycling when wild type cells remained arrested. Thus, deregulation of ALG7 interferes with cell cycle arrest by preventing a sustained downregulation of CLN1 and CLN2 mRNAs. These results provide a molecular insight into the role of ALG7, and protein N-glycosylation in general, in proliferation.

The dual role of mRNA half-lives in the expression of the yeast ALG7 gene.

The yeast ALG7 gene functions by initiating the synthesis of the dolichol-linked oligosaccharide precursor and plays an important role in the control of protein N-glycosylation. The levels of ALG7 multiple transcripts are modulated by the physiological status of the cell and environmental cues, and deregulation of their abundance is deleterious to several cellular functions. Since ALG7 mRNAs are unstable, we investigated the role of these transcripts' half-lives in determining their steady-state levels. Using a temperature-sensitive RNA polymerase II mutant, we demonstrate that increased stability was the primary determinant of higher ALG7 mRNA abundance in response to glucose limitation or treatment with tunicamycin. In contrast, at the G1/G0 transition point, changes in the decay rates were inversely related to ALG7 transcript accumulation: the decreased abundance of ALG7 mRNAs following exit from the mitotic cycle was associated with lengthening of the decay rates, while their increased accumulation after growth stimulation correlated with decreased stability. This suggests that, depending on the circumstance, mRNA half-lives can either directly determine the level of ALG7 transcript accumulation or oppose regulatory changes at other control levels.

Proliferation-dependent differential regulation of the dolichol pathway genes in Saccharomyces cerevisiae.

The dolichol pathway serves in the synthesis of the dolichol-linked oligosaccharide precursor for protein N-glycosylation. Recently, we reported that mRNAs of genes that function at the early steps in the dolichol pathway in yeast, ALG7, ALG1 and ALG2, were co-ordinately induced following growth stimulation of G0-arrested cells in a manner similar to that of the transcripts of the early growth response genes (Kukuruzinska, M.A. and Lennon, K. Glycobiology, 4, 437-443, 1994). To determine whether the entire dolichol pathway was co-ordinately regulated with growth, we examined the expression of genes functioning late in the pathway, including two genes encoding oligosaccharyltransferase subunits, at two critical control points in the G1 phase of cell cycle: G0/G1 and START. We show that early in G1, at the G0/G1 transition point, the late ALG genes and the two oligosaccharyltransferase-encoding genes examined were regulated co-ordinately with the early ALG genes: they were downregulated upon exit from the mitotic cell cycle into G0, and they were induced following growth stimulation in the absence of de novo protein synthesis. All the dolichol pathway genes produced transcripts with short half-lives that were rapidly stabilized in the presence of cycloheximide. In contrast, cell division arrest late in G1, at START, was accompanied by a selective downregulation of only the first dolichol pathway gene, ALG7, and not of the genes functioning later in the pathway. These results indicate that, depending on their position in G1, cells either co-ordinately or differentially regulate the dolichol pathway genes.

Expression of the first N-glycosylation gene in the dolichol pathway, ALG7, is regulated at two major control points in the G1 phase of the Saccharomyces cerevisiae cell cycle.

The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene. To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle. We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of alpha-factor. ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START. In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by alpha-factor treatment later in G1. Importantly, deregulated expression of ALG7 resulted in an aberrant alpha-factor response. Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START.

Diminished activity of the first N-glycosylation enzyme, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), gives rise to mutant phenotypes in yeast.

The enzyme which initiates the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), is encoded by the ALG7 gene. Essential for viability, ALG7 has been evolutionarily conserved and shown to be involved in a variety of functions. ALG7 is an early growth-response gene in yeast, and downregulation of ALG7 expression results in diminished N-glycosylation and secretion of Xenopus oocyte proteins. We have now investigated the consequences of diminished GPT activity in yeast using mutant ALG7 genes with deletions in the 3' untranslated region (3' UTR). We show that a 2.5- to 4-fold reduction in GPT activity gave rise to distinct phenotypes, whose severity was inversely related to the level of GPT activity. These phenotypes included hypersensitivity to tunicamycin, enlarged cell size, extensive aggregation, lack of a typical stationary (G0) arrest, and defective spore germination. We conclude that yeast cells are sensitive to GPT dosage, and that attenuation of GPT activity interferes with various functions in the yeast life cycle.

Developmental regulation and tissue-specific expression of hamster dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT).

The first enzyme in the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-phosphate transferase, GPT, has been implicated in the development of a wide variety of eukaryotes. GPT is encoded by ALG7, an early growth-response gene, whose expression has been shown to affect the extent of N-glycosylation and secretion of proteins. To initiate the molecular characterization of ALG7 involvement in mammalian growth and differentiation, we have used the postnatally developing hamster submandibular gland (SMG) as an experimental paradigm. In this study we report that the ALG7 gene was differentially expressed during postnatal development and in terminally differentiated adult tissues. Throughout development, GPT activity paralleled ALG7 mRNA levels, suggesting that the amount of functional enzyme was determined by modulation of transcript abundance.

Growth-related coordinate regulation of the early N-glycosylation genes in yeast.

The Saccharomyces cerevisiae ALG7, ALG1 and ALG2 genes, whose products function early in the dolichol pathway of protein N-glycosylation, are essential for cell viability, and perturbation in their expression causes G1-specific cell cycle arrest. Here, we show that expression of the ALG7, ALG1 and ALG2 genes is coordinately regulated at the G0/G1 transition point in the yeast life cycle. Carbon starvation, which induces cells to exit from the G1 stage of the mitotic cycle into G0, resulted in a time-dependent decrease in the levels of the early ALG genes' mRNAs. Accordingly, addition of glucose, which stimulates the G0-arrested cells to resume proliferation, resulted in a rapid induction of their mRNAs. Cycloheximide alone also induced the early ALG transcripts, albeit to a much lower extent than glucose. Simultaneous addition of glucose and cycloheximide caused superinduction of these mRNAs, indicating that more than one control level was involved in their activation. Consistent with this, rapid degradation of ALG7, ALG1 and ALG2 mRNAs was completely abolished in the presence of cycloheximide. These data suggest that in yeast, the early N-glycosylation genes are regulated in a manner similar to that of the early growth-response genes.

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: the alg2 mutation.

In the yeast Saccharomyces cerevisiae, the alg2 mutation causes temperature-sensitive growth and abnormal accumulation of the lipid-linked oligosaccharide Man2GlcNAc2-PP-Dol (Jackson et al., Arch. Biochem. Biophys., 272, 203-209, 1989; Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). A gene having the function and genomic location of ALG2 was cloned from libraries based on the multicopy plasmid YEp24 and on the centromere plasmid YCp50. Alg2 mutants transformed with plasmids containing ALG2 regained the capacity to grow and to synthesize lipid-linked oligosaccharides normally at the previously non-permissive temperature. ALG2 was essential for viability in haploid and diploid yeast. The ALG2 gene was transcribed into a single mRNA of 1.7 kb in size. The stability of ALG2 mRNA, assessed after thermal inactivation of RNA polymerase II in an rpb1-1 mutant (Herrick et al., Mol. Cell. Biol., 10, 2269-2284, 1990) was very low, with a t1/2 of < 5 min. The ALG2 transcript accumulation was growth dependent, and it was at least an order of magnitude lower in stationary phase cells compared to exponentially growing cells. The putative translation product of ALG2 contained a potential dolichol recognition domain similar to that found in all three glycosyltransferases of the lipid-linked pathway that have been sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein glycosylation in yeast: transcript heterogeneity of the ALG7 gene.

The first enzyme in the lipid-linked oligosaccharide biosynthetic pathway, UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (UDP-N-acetyl-D-glucosamine:dolichyl-phosphate-N-acetyl- D-glucosaminephosphotransferase, EC 2.7.8.15), is encoded by the ALG7 gene. We show that this gene is essential for cell growth, since a null mutation constructed with standard gene disruption techniques results in cell lethality. The ALG7 gene is transcribed into two major messages, approximately 1.38 and 1.56 kilobase pairs (kbp) in size, and this heterogeneity has been mapped to the 3' untranslated region. Two sets of tripartite sequences implicated in transcription termination begin 15 bp and 256 bp past the translation stop codon, TGA. The ratios of the two major transcripts change with gene dosage, with the longer mRNA becoming more abundant in cells containing higher levels of the ALG7 gene. Changes in transcript ratios are also observed in mutants defective in lipid-linked sugar-donor biosynthesis. In addition, there is 5' heterogeneity in the ALG7 mRNAs. The transcripts start at four initiation sites located within a 20-bp region. Two potentially functional TATA elements have been identified at positions -157 and -139, which may be involved in initiation from multiple sites. These features point to numerous factors that may be involved in the regulation of the expression of the ALG7 gene.