Howard C. Crawford

Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse

To evaluate the role of oncogenic RAS mutations in pancreatic tumorigenesis, we directed endogenous expression of KRAS(G12D) to progenitor cells of the mouse pancreas. We find that physiological levels of Kras(G12D) induce ductal lesions that recapitulate the full spectrum of human pancreatic intraepithelial neoplasias (PanINs), putative precursors to invasive pancreatic cancer. The PanINs are highly proliferative, show evidence of histological progression, and activate signaling pathways normally quiescent in ductal epithelium, suggesting potential therapeutic and chemopreventive targets for the cognate human condition. At low frequency, these lesions also progress spontaneously to invasive and metastatic adenocarcinomas, establishing PanINs as definitive precursors to the invasive disease. Finally, mice with PanINs have an identifiable serum proteomic signature, suggesting a means of detecting the preinvasive state in patients.

The metalloproteinase matrilysin is a target of β-catenin transactivation in intestinal tumors

Matrilysin is a matrix metalloproteinase expressed in the tumor cells of greater than 80% of intestinal adenomas. The majority of these intestinal tumors are associated with the accumulation of β-catenin, a component of the cadherin adhesion complex and, through its association with the T Cell Factor (Tcf) DNA binding proteins, a regulator in the Wnt signal transduction pathway. In murine intestinal tumors, matrilysin transcripts show striking overlap with the accumulation of β-catenin protein. The matrilysin promoter is upregulated as much as 12-fold by β-catenin in colon tumor cell lines in a manner inversely proportional to the endogenous levels of β-catenin/Tcf complex and is dependent upon a single optimal Tcf-4 recognition site. Coexpression of the E-cadherin cytoplasmic domain blocked this induction and reduced basal promoter activity in every colon cancer cell line tested. Inactivation of the Tcf binding site increased promoter activity and overexpression of the Tcf factor, LEF-1, significantly downregulated matrilysin promoter activity, suggesting that β-catenin transactivates the matrilysin promoter by virtue of its ability to abrogate Tcf-mediated repression. Because genetic ablation of matrilysin decreases tumor formation in multiple intestinal neoplasia (Min) mice, we propose that regulation of matrilysin production by β-catenin accumulation is a contributing factor to intestinal tumorigenesis.

Inhibition of RhoA by p120 catenin

RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell–cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.

Matrix metalloproteinase-7–dependent release of tumor necrosis factor-α in a model of herniated disc resorption

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.

The p120ctn-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides

The p120(ctn)-binding partner Kaiso is a new member of the POZ-zinc finger family of transcription factors implicated in development and cancer. To understand the role of Kaiso in gene regulation and p120(ctn)-mediated signaling and adhesion, we sought to identify Kaiso-specific DNA binding sequences and potential target genes. Here we demonstrate that Kaiso is a dual specificity DNA-binding protein that recognizes the specific consensus sequence TCCTGCNA as well as methyl-CpG dinucleotides. A minimal core sequence CTGCNA was identified as sufficient for Kaiso binding. Two copies of the Kaiso-binding site are present in the human and murine matrilysin promoters, implicating matrilysin as a candidate target gene for Kaiso. In electrophoretic mobility shift assays, matrilysin promoter-derived oligonucleotide probes formed a complex with GST-Kaiso fusion proteins possessing the zinc finger domain but not with fusion proteins lacking the zinc fingers. We further determined that only Kaiso zinc fingers 2 and 3 were necessary and sufficient for sequence-specific DNA binding. Interestingly, Kaiso also possesses a methyl-CpG-dependent DNA-binding activity distinct from its sequence-specific DNA binding. However, Kaiso has a higher affinity for the TCCTGCNA consensus than for the methyl-CpG sites. Furthermore, the DNA-binding ability of Kaiso with either recognition site was inhibited by p120(ctn). Kaiso thus appears to have two modes of DNA binding and transcriptional repression, both of which may be modulated by its interaction with the adhesion cofactor p120(ctn).

Adipogenesis is inhibited by brief, daily exposure to high-frequency, extremely low-magnitude mechanical signals

Obesity, a global pandemic that debilitates millions of people and burdens society with tens of billions of dollars in health care costs, is deterred by exercise. Although it is presumed that the more strenuous a physical challenge the more effective it will be in the suppression of adiposity, here it is shown that 15 weeks of brief, daily exposure to high-frequency mechanical signals, induced at a magnitude well below that which would arise during walking, inhibited adipogenesis by 27% in C57BL/6J mice. The mechanical signal also reduced key risk factors in the onset of type II diabetes, nonesterified free fatty acid and triglyceride content in the liver, by 43% and 39%, respectively. Over 9 weeks, these same signals suppressed fat production by 22% in the C3H.B6–6T congenic mouse strain that exhibits accelerated age-related changes in body composition. In an effort to understand the means by which fat production was inhibited, irradiated mice receiving bone marrow transplants from heterozygous GFP+ mice revealed that 6 weeks of these low-magnitude mechanical signals reduced the commitment of mesenchymal stem cell differentiation into adipocytes by 19%, indicating that formation of adipose tissue in these models was deterred by a marked reduction in stem cell adipogenesis. Translated to the human, this may represent the basis for the nonpharmacologic prevention of obesity and its sequelae, achieved through developmental, rather than metabolic, pathways.

The PEA3 subfamily of Ets transcription factors synergizes with β-catenin-LEF-1 to activate matrilysin transcription in intestinal tumors

ABSTRACT The matrix metalloproteinase matrilysin (MMP-7) is expressed in the tumor cells of a majority of mouse intestinal and human colonic adenomas. We showed previously that matrilysin is a target gene of β-catenin–Tcf, the transcription factor complex whose activity is thought to play a crucial role in the initiation of intestinal tumorigenesis. Here we report that overexpression of a stable mutant form of β-catenin alone was not sufficient to effect expression of luciferase from a matrilysin promoter-luciferase reporter plasmid. However, cotransfection of the reporter with an expression vector encoding the PEA3 Ets transcription factor, or its close relatives ER81 and ERM, increased luciferase expression and rendered the promoter responsive to β-catenin–LEF-1 as well as to the AP-1 protein c-Jun. Other Ets proteins could not substitute for the PEA3 subfamily. Luciferase activity was induced up to 250-fold when PEA3, c-Jun, β-catenin, and LEF-1 were coexpressed. This combination of transcription factors was also sufficient to induce expression of the endogenous matrilysin gene. Furthermore, all matrilysin-expressing benign intestinal tumors of the Min mouse expressed a member of the PEA3 subfamily, as did all human colon tumor cell lines examined. These data suggest that the expression of members of the PEA3 subfamily, in conjunction with the accumulation of β-catenin in these tumors, leads to coordinate upregulation of matrilysin gene transcription, contributing to gastrointestinal tumorigenesis.

Matrix metalloproteinase-3–dependent generation of a macrophage chemoattractant in a model of herniated disc resorption

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

Loss of FOXA1/2 is essential for the epithelial-to-mesenchymal transition in pancreatic cancer.

FOXA1 and FOXA2, members of the forkhead transcription factor family, are critical for epithelial differentiation in many endoderm-derived organs, including the pancreas. However, their role in tumor progression is largely unknown. Here, we identified FOXA1 and FOXA2 as important antagonists of the epithelial-to-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDA) through their positive regulation of E-cadherin and maintenance of the epithelial phenotype. In human PDA samples, FOXA1/2 are expressed in all epithelia from normal to well-differentiated cancer cells, but are lost in undifferentiated cancer cells. In PDA cell lines, FOXA1/2 expression is consistently suppressed in experimental EMT models and RNAi silencing of FOXA1/2 alone is sufficient to induce EMT. Conversely, ectopic FOXA1/2 expression can potently neutralize several EMT-related E-cadherin repressive mechanisms. Finally, ectopic FOXA2 expression could reactivate E-cadherin expression in a PDA cell line with extensive promoter hypermethylation. In fact, demethylation-mediated reactivation of E-cadherin expression in these cells required concurrent reactivation of endogenous FOXA2 expression. We conclude that suppression of FOXA1/2 expression is both necessary and sufficient for EMT during PDA malignant progression.

PEA3 Is Up-regulated in Response to Wnt1 and Activates the Expression of Cyclooxygenase-2

The inducible prostaglandin synthase cyclooxygenase-2 (COX-2) is aberrantly expressed in intestinal tumors resulting from APC mutation, and is also transcriptionally up-regulated in mouse mammary epithelial cells in response toWnt1 expression. β-Catenin stabilization is a consequence of both APC mutation and Wnt signaling. We have previously observed coordinate regulation of the matrilysin promoter by β-catenin and Ets family transcription factors of the PEA3 subfamily. Here we show that while β-catenin only weakly activates theCOX-2 promoter, PEA3 family transcription factors are potent activators of COX-2 transcription. Consistent with this, PEA3 is up-regulated in Wnt1-expressing mouse mammary epithelial cells, and PEA3 factors are highly expressed in tumors from Wnt1 transgenic mice, in whichCox-2 is also up-regulated. Promoter mapping experiments suggest that the NF-IL6 site in the COX-2 promoter is important for mediating PEA3 responsiveness. The NF-IL6 site is also important for COX-2 transcription in some colorectal cancer lines (Shao, J., Sheng, H., Inoue, H., Morrow, J. D., and DuBois, R. N. (2000) J. Biol. Chem. 275, 33951–33956), and PEA3 factors are highly expressed in colorectal cancer cell lines. Therefore, we speculate that PEA3 factors may contribute to the up-regulation of COX-2 expression resulting from bothAPC mutation and Wnt1 expression.