RSK Activation of Translation Factor eIF4B Drives Abnormal Increases of Laminin γ2 and MYC Protein during Neoplastic Progression to Squamous Cell Carcinoma

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5′ untranslated regions (5′-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5′-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC

β-Catenin–induced melanoma growth requires the downstream target Microphthalmia-associated transcription factor

The transcription factor Microphthalmia-associated transcription factor (MITF) is a lineage-determination factor, which modulates melanocyte differentiation and pigmentation. MITF was recently shown to reside downstream of the canonical Wnt pathway during melanocyte differentiation from pluripotent neural crest cells in zebrafish as well as in mammalian melanocyte lineage cells. Although expression of many melanocytic/pigmentation markers is lost in human melanoma, MITF expression remains intact, even in unpigmented tumors, suggesting a role for MITF beyond its role in differentiation. A significant fraction of primary human melanomas exhibit deregulation (via aberrant nuclear accumulation) of β-catenin, leading us to examine its role in melanoma growth and survival. Here, we show that β-catenin is a potent mediator of growth for melanoma cells in a manner dependent on its downstream target MITF. Moreover, suppression of melanoma clonogenic growth by disruption of β-catenin–T-cell transcription factor/LEF is rescued by constitutive MITF. This rescue occurs largely through a prosurvival mechanism. Thus, β-catenin regulation of MITF expression represents a tissue-restricted pathway that significantly influences the growth and survival behavior of this notoriously treatment-resistant neoplasm

Dual Suppression of the Cyclin-Dependent Kinase Inhibitors CDKN2C and CDKN1A in Human Melanoma

Resistance to BRAFV600E inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAFV600E inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF V600E or NRAS G12D and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAFV600E promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF V600E or NRAS G12D-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAFV600E- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAFV600E- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 nu mice (P = .03 when mean tumor volume at day 13 was compared for BRAFV600E inhibitor vs BRAFV600E inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4–8 mice per group)

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD + regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD + /NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. Keyword: PIK3CA; 2OG; OGDH; TCA cycle; glycolysisDamon Runyon Cancer Research Foundation (HHMI Fellowship

Tuning transcription factor availability through acetylation-mediated genomic redistribution

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability

Chromatin Structure: Nucleosome Formation and Positioning

In all eukaryotic cells the DNA is complexed with a group of highly basic histone proteins forming nucleosomes and higher order chromatin structures. In the nucleosome the DNA is tightly wrapped, almost two turns, around an octamer of histone proteins. The octamer in turn is built as a tripartite assembly of a (H3/H4)2-tetramer flanked by two (H2A/H2B)-dimers. The tripartite nature exhibits an interesting feature upon nucleosome formation. The nucleosomal wrapping about the tetramer complex is stabilised energetically four-fold by the addition of the dimers. In addition, the histone octamer is designed to bind virtually all sequences that exist in the genome, but intrinsic properties of the DNA, such as curvature and flexibility, modulate the association of a particular stretch with the histone octamer. The protruding histone terminal tails also contribute to nucleosome stability. The tails help folding on the mononucleosomal level of intrinsically static DNA, but not for flexible DNA. On the chromatin level the tails mediate folding interactions to approximately 800cal/mol which is in the order of magnitude for sequence dependent variations of nucleosome positioning. In addition, we have also found that acetylation of the histone tails facilitate nucleosome formation. Using an in vitro selection strategy, sequences from the mouse genome were identified that exhibited a high affinity for histone octamers. These were categorised by their sequence context and were found to fall into three major groups; highly flexible due to abundance of TG/CA dinucleotides, intrinsically curved arising from phased A-tracts and last, a group of sequences with no obvious characteristics. One minor group of (TATAAACGCC)-repeat sequences were found to have the highest affinity in nucleosome formation from genomic material, which is about 350-fold higher than average nucleosomal DNA, largely dependent on their 25-fold higher flexibility relative to average DNA. In addition, extensive TGGA-repeats were identified by a selection study for nucleosome refractory sequences from synthetic DNA. These repeats were found to be abundant constituents of the mouse genome and predominately located to five discrete locations on mouse metaphase chromosomes. Hybridisation to human chromosomes revealed that these repeats are located in telomeric positions

Chromatin Structure: Nucleosome Formation and Positioning

In all eukaryotic cells the DNA is complexed with a group of highly basic histone proteins forming nucleosomes and higher order chromatin structures. In the nucleosome the DNA is tightly wrapped, almost two turns, around an octamer of histone proteins. The octamer in turn is built as a tripartite assembly of a (H3/H4)2-tetramer flanked by two (H2A/H2B)-dimers. The tripartite nature exhibits an interesting feature upon nucleosome formation. The nucleosomal wrapping about the tetramer complex is stabilised energetically four-fold by the addition of the dimers. In addition, the histone octamer is designed to bind virtually all sequences that exist in the genome, but intrinsic properties of the DNA, such as curvature and flexibility, modulate the association of a particular stretch with the histone octamer. The protruding histone terminal tails also contribute to nucleosome stability. The tails help folding on the mononucleosomal level of intrinsically static DNA, but not for flexible DNA. On the chromatin level the tails mediate folding interactions to approximately 800cal/mol which is in the order of magnitude for sequence dependent variations of nucleosome positioning. In addition, we have also found that acetylation of the histone tails facilitate nucleosome formation. Using an in vitro selection strategy, sequences from the mouse genome were identified that exhibited a high affinity for histone octamers. These were categorised by their sequence context and were found to fall into three major groups; highly flexible due to abundance of TG/CA dinucleotides, intrinsically curved arising from phased A-tracts and last, a group of sequences with no obvious characteristics. One minor group of (TATAAACGCC)-repeat sequences were found to have the highest affinity in nucleosome formation from genomic material, which is about 350-fold higher than average nucleosomal DNA, largely dependent on their 25-fold higher flexibility relative to average DNA. In addition, extensive TGGA-repeats were identified by a selection study for nucleosome refractory sequences from synthetic DNA. These repeats were found to be abundant constituents of the mouse genome and predominately located to five discrete locations on mouse metaphase chromosomes. Hybridisation to human chromosomes revealed that these repeats are located in telomeric positions

TGGA-repeats impair nucleosome formation

9C, SE-413 90, Go Èteborg Sweden Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in genome condensation, and in¯uence transcription and replication. Properties of the DNA sequence, such as curvature and¯exibility, direct the location of nucleosomes. DNA sequences that position nucleosomes have been identi®ed and rules that govern their properties have been formulated. However, DNA sequences that are refractory to nucleosome formation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequences by selecting those that refrain from nucleosome formation from a large pool of synthetic DNA fragments with a central region of 146 random base-pairs ®tted with adapters for PCR ampli®cation. These were used for in vitro salt-induced reconstitution of nucleosomes under thermodynamic equilibrium conditions. Fragments that did not form nucleosomes were puri®ed, ampli®ed by PCR, and the reconstitution was repeated. After 17 rounds of negative selection, the material was highly enriched in sequences reluctant to form nucleosomes. Cloning and sequencing revealed that 35% of the molecules had long repeats of TGGA, and their af®nity for histone octamers was about half that of average DNA