Marian Waterman - Academia.edu (original) (raw)

Papers by Marian Waterman

Nucleic Acids Research, May 1, 2000

Lymphoid Enhancer Factor-1 (LEF-1) is a member of a family of transcription factors that function... more Lymphoid Enhancer Factor-1 (LEF-1) is a member of a family of transcription factors that function as downstream mediators of the Wnt signal transduction pathway. In the absence of Wnt signals, specific LEF/TCF isoforms repress rather than activate gene targets through recruitment of the co-repressor CtBP. Characterization of the full-length human LEF-1 gene locus and its complete set of mRNA products shows that this family member exists as a unique set of alternatively spliced isoforms; none are homologous to TCF-1E/TCF-4E. Therefore LEF-1 is distinct from its TCF family members in that it cannot engage in activities specific to this isoform such as recruitment of the co-repressor CtBP. Expression of alternatively spliced LEF-1 isoforms are driven by a promoter that is highly active in lymphocyte cell lines. Transcription initiates within a TATA-less core promoter region that contains consensus binding sites for Sp1, an E box, an Initiator element and a LEF/TCF binding site, all juxtaposed to the start sites of transcription. The promoter is most active in a B lymphocyte cell line (Raji) in which the endogenous LEF-1 gene is silent, suggesting that the promoter region is actively repressed by a silencing mechanism.

Integrative Biology, 2014

Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic pro... more Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic process include: the formation of new blood vessels, the initiation of epithelial-mesenchymal transition (EMT), and the mobilization of tumor cells into the circulation. There are ongoing efforts to replicate the physiological landscape of human tumor tissue using three-dimensional in vitro culture models; however, few systems are able to capture the full range of authentic, complex in vivo events such as neovascularization and intravasation. Here we introduce the Prevascularized Tumor (PVT) model to investigate early events of solid tumor progression. PVT spheroids are composed of endothelial and tumor cells, and are embedded in a fibrin matrix containing fibroblasts. The PVT model facilitates two mechanisms of vessel formation: robust sprouting angiogenesis into the matrix, and contiguous vascularization within the spheroid. Furthermore, the PVT model enables the intravasation of tumor cells that is enhanced under low oxygen conditions and is also dependent on the key EMT transcription factor Slug. The PVT model provides a significant advance in the mimicry of human tumors in vitro and may improve investigation and targeting of events in the metastatic process.

Blood, Nov 1, 2006

The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple gen... more The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of Bcr-Abl in dysregulating translation. Our group has previously documented the effects of Bcr-Abl on key regulators of cap-dependent translation and the role that this mechanism plays in transformation (Ly et al, Cancer Research, 2003; Prabhu et al., Oncogene, in press). Here we describe a novel form of translational control by Bcr-Abl. Specifically, we show how Bcr-Abl regulates cap-independent translation of Lymphoid Enhancer Factor-1 (LEF-1) via a bona fide internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of LEF1. Lymphoid Enhancer Factor-1 (LEF-1), a transcription factor that mediates Wnt signals via interaction with β-catenin, is often expressed in cancers derived from aberrant Wnt signaling. Lately, it has been reported that LEF1 transcripts are elevated in CML. We examined LEF-1 expression in primary CML cells and cell lines (K562 and Ba/F3-Bcr-Abl) and show that LEF-1 protein is detected in all patient-derived cells. Treatment of these cells with the Bcr-Abl imatinib mesylate (imatinib) inhibits LEF-1 expression in imatinib-sensitive cancers, but not in cancers that exhibit clinical resistance even though such cancers express imatinib-sensitive Bcr-Abl. For those cancers that are sensitive, inhibition of Bcr-Abl has a partial effect on LEF1 mRNA levels, and a significant effect on LEF-1 protein levels. LEF-1 protein is produced via two IRESs in it’s 5′ UTR. IRES-driven translation of LEF-1 was highly sensitive to Bcr-Abl as treatment with imatinib reduced IRES activity 5 fold. Transfection of CML cells with dicistronic mRNAs suggests that Bcr-Abl stimulates LEF-1 protein production through steps in the nucleus and cytoplasm. We propose that, in addition to its strong effects on cap-dependent translation in CML, Bcr-Abl is an important regulator of alternative translation pathways. mRNAs that are translated via IRES-dependent mechanisms are particularly relevant to cancer since they encode proteins that regulate cell proliferation and survival. Together, these observations underscore the important role which dysregulated translation plays in transformation, and suggest novel approaches with which to counteract the transforming properties of Bcr-Abl.

Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting... more Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting cells such as the stroma and vasculature, mechanical factors, such as interstitial flow and extracellular matrix and aspects of the tumor mass itself, including shape. However, current tumor growth models, including xenograft models, 2D and/or simplistic 3D cultures, are unable to address these interactions in a high throughput human-derived system. We have developed a novel microfluidic platform that combines human derived perfused microvessels, stroma, and interstitial flow with 3D culture. This platform was used to quantitatively compare the role of these microenvironmental factors on tumor growth. Methods: A microfluidic device was fabricated consisting of two supply channels on either side of a central tissue compartment. The inner stromal compartment consists of normal human fibroblasts (NHLFs) and GFP-labeled human colorectal adenocarcinoma tumor cells, SW620, seeded in a fibrin matrix. To simulate a vasculogenic-like process, human cord blood endothelial colony forming cells endothelial cells (ECFC-ECs) were distributed throughout the stromal channel with the fibroblasts and tumor cells. Tumor growth rate and area was compared across day, interstitial flow rates and tumor shape (fractal dimension, perimeter to area) with ANOVA. Results: Cell viability within the device was maintained under interstitial flow conditions for a period of 21 days. Within one week of culture, microvessel formation and significant tumor growth into spheroids (n=636) were observed. On average, tumor growth rate was 26% ±62% per day with the highest growth rates observed on the first days. By day 7, many tumor masses had died off, with 2-3 large, fast growing tumors remaining per chamber. Highest tumor growth rates and areas were observed in tumor masses with a characteristic morphology of high perimeter to area and lower cohesion. Interstitial flow rates ranging from essentially static to supraphysiologic were generated. Differences in tumor growth rates were not statistically significant across chambers with different mean flow rates. To demonstrate intraluminal flow within the vascular network, fluorescently labeled dextran (40, 70, and 150 kDa) was introduced into the microfluidic lines. Dextran was retained in the vessel network, and showed tumor cells residing in the intraluminal space of the formed vasculature. By day 14, the network eroded, as the tumor masses overgrew and encompassed more than 60% of the chamber volume. Discussion: We have developed a novel 3D microfluidic system of the tumor microenvironment that features perfused capillaries and controlled interstitial flow. Tumor growth was affected by tumor characteristic shape in this model though interstitial flow appeared to play a lesser role. Vascular development was observed and its interaction with tumor growth will be analyzed in future work. Citation Format: Luis F. Alonzo, Claire J. Robertson, Monica L. Moya, Marian L. Waterman, Christopher C. Hughes, Steven C. George. Recapitulating the microenvironment in vitro for comparative study of factors affecting tumor growth and vascularization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3930. doi:10.1158/1538-7445.AM2014-3930

Cell, Dec 1, 2012

Two new studies reveal ways in which the Wnt pathway commandeers Hippo components for signaling. ... more Two new studies reveal ways in which the Wnt pathway commandeers Hippo components for signaling. Azzolin et al. show how the Hippo transcription factor TAZ mediates Wnt signals, and Rosenbluh et al. show how b-catenin and YAP1 form a kinase-regulated complex with transcription factor TBX5.

Neuroscience, May 1, 2015

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerv... more Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via β-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and β-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and β-catenin levels. Moreover, a novel Wnt/β-catenin transgenic reporter mouse line was utilized to

Science, Jul 19, 1985

In experiments designed to study the mechanism by which peptide hormones binding to their plasma ... more In experiments designed to study the mechanism by which peptide hormones binding to their plasma membrane receptors stimulate the expression of specific genes, the transcription of two neuroendocrine genes, prolactin and growth hormone, was analyzed in a rat pituitary cell line. The results showed that cyclic adenosine monophosphate (cyclic AMP) stimulates the transcription of discrete subsets of eukaryotic genes by at least two independent molecular mechanisms. Cyclic AMP stimulated growth hormone gene transcription and phosphorylation of a 19,000-dalton nuclear protein; this appears to reflect direct nuclear actions of the cyclic AMP-dependent protein kinase. In contrast, the stimulation by cyclic AMP of prolactin gene transcription appears to reflect activation of a discrete calcium-dependent event.

Differentiation, Sep 1, 2012

Wnt ligands are secreted morphogens that control multiple developmental processes during embryoge... more Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.

The FASEB Journal, Apr 1, 2010

Current Opinion in Gastroenterology, 2002

Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is inv... more Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development. Mutation of midstream components can activate the pathway, making it independent of Wnt ligands and maintaining constant pressure to change target gene expression. The transcription factors that connect the pathway to target genes are members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family. The genes for two members of this family, TCF 7 and LEF 1, produce full-length forms that mediate Wnt signals and truncated dominant negative forms that limit Wnt signals and may function as growth suppressors. Results from studies of their expression in colon cancer suggests that because Wnt-linked cancers progress to malignancy, there may be a strengthening of the Wnt signal by selective expression of the activating forms of LEF/TCFs and a bias against suppressing, truncated forms.

The EMBO Journal, Mar 3, 2014

Wnt signaling activates target genes by promoting association of the co-activator b-catenin with ... more Wnt signaling activates target genes by promoting association of the co-activator b-catenin with TCF/LEF transcription factors. In the absence of b-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between b-catenin and TLE for TCFs as part of an activation-repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

Springer eBooks, Dec 14, 2013

There has been increasing evidence of the critical effects of microenvironmental influence on tum... more There has been increasing evidence of the critical effects of microenvironmental influence on tumor growth and metastasis. In this report, we extend a multispecies continuum model of solid tumor growth to include interaction of the tumor with its microenvironment. This new model, which incorporates reported interactions between tumor-and stroma-derived chemical signals, predicts a nonlinear response to host factors: increased growth and asymmetry of the tumor at low levels of stromal fibroblast-produced Hepatocyte Growth Factor / Scatter Factor (HGF/SF), and reduced growth at high levels.We test the model predictions using colon cancer initiating cell (CCIC) spheroids grown in media in varying concentrations of HGF. The experiments show qualitatively similar behavior to the model predictions. We plan to use the experimental studies to calibrate the mathematical model, and to use the mathematical model to make predictions regarding tumor behavior in order to guide future experimental studies.

Biophysical Journal, 2013

Molecular and Cellular Biology, Sep 15, 2012

Genes & Development, Apr 1, 1991

The human T cell-specific transcription factor TCF-la plays a key role in the tissue-specific act... more The human T cell-specific transcription factor TCF-la plays a key role in the tissue-specific activation of the T cell receptor (TCR) Ca enhancer and binds to pyrimidine-rich elements (5'-PyCTTTG-3') present in a variety of other T cell-specific control regions. Using amino acid sequence information derived from the DNA affinity-purified protein, we have now isolated cDNA clones encoding TCF-la. The TCF-Ia cDNA contains a single 68-amino-acid domain that is homologous to a region conserved among high-mobility group (HMG) and nonhistone chromosomal proteins. Expression of full-length and mutant cDNA clones in bacteria reveal that the single HMG motif, which is predicted to contain two extended a-helical segments, is sufficient to direct the sequence-specific binding of TCF-lc~ to DNA. Northern blot experiments demonstrate further that TCF-I,~ mRNA is highly tissue specific, found primarily in the thymus or T cell lines. The immature CEM T cell line expresses relatively low levels of TCF-la mRNA, which are increased upon activation of these cells by phorbol esters. Interestingly, the cloned TCF-I¢~ protein is a potent transcriptional activator of the human TCRa enhancer in nonlymphoid cell lines, whereas the activity of the endogenous protein in T cell lines is strongly dependent on an additional T cell-specific protein that interacts with the core enhancer. TCF-let is currently unique among the newly emerging family of DNA-binding regulatory proteins that share the HMG motif in that it is a highly tissue-specific RNA polymerase II transcription factor.

Cold Spring Harbor Perspectives in Biology, Sep 28, 2012

Wingless signaling throughout metazoans. TCF/LEFs are multifunctional proteins that use their seq... more Wingless signaling throughout metazoans. TCF/LEFs are multifunctional proteins that use their sequence-specific DNA-binding and context-dependent interactions to specify which genes will be regulated by Wnts. Much of the work to define their actions has focused on their ability to repress target gene expression when Wnt signals are absent and to recruit b-catenin to target genes for activation when Wnts are present. Recent advances have highlighted how these on/off actions are regulated by Wnt signals and stabilized b-catenin. In contrast to invertebrates, which typically contain one TCF/LEF protein that can both activate and repress Wnt targets, gene duplication and isoform complexity of the family in vertebrates have led to specialization, in which individual TCF/LEF isoforms have distinct activities.