TRIM29 (ATDC) exhibits a contextual function in cancer, but seems to exert a tumor-suppressor role in breast cancer. Here, we show that TRIM29 is often silenced in primary breast tumors and cultured tumor cells as a result of aberrant gene hypermethylation. RNAi-mediated silencing of TRIM29 in breast tumor cells increased their motility, invasiveness, and proliferation in a manner associated with increased expression of mesenchymal markers (N-cadherin and vimentin), decreased expression of epithelial markers (E-cadherin and EpCAM), and increased expression and activity of the oncogenic transcription factor TWIST1, an important driver of the epithelial–mesenchymal transition (EMT). Functional investigations revealed an inverse relationship in the expression of TRIM29 and TWIST1, suggesting the existence of a negative regulatory feedback loop. In support of this relationship, we found that TWIST1 inhibited TRIM29 promoter activity through direct binding to a region containing a cluster of consensus E-box elements, arguing that TWIST1 transcriptionally represses TRIM29 expression. Analysis of a public breast cancer gene-expression database indicated that reduced TRIM29 expression was associated with reduced relapse-free survival, increased tumor size, grade, and metastatic characteristics. Taken together, our results suggest that TRIM29 acts as a tumor suppressor in breast cancer through its ability to inhibit TWIST1 and suppress EMT. Cancer Res; 74(17); 4875–87. ©2014 AACR.

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181–19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6–7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and β-galactosidase, as well as several other approaches. From these studies, we conclude that the NH₂ terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal ∼25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHAS-related domains.

Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B) is caused by a deficiency in the lysosomal enzyme N-acetyl-glucosaminidase (Naglu). Children with MPS IIIB develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death. The murine model of MPS IIIB demonstrates lysosomal distention in multiple tissues, a shortened life span, and behavioral changes. To more thoroughly assess MPS IIIB in mice, alterations in circadian rhythm, activity level, motor function, vision, and hearing were tested. The suprachiasmatic nucleus (SCN) developed pathologic changes and locomotor analysis showed that MPS IIIB mice start their daily activity later and have a lower proportion of activity during the night than wild-type controls. Rotarod assessment of motor function revealed a progressive inability to coordinate movement in a rocking paradigm. Purkinje cell counts were significantly reduced in the MPS IIIB animals compared to age matched controls. By electroretinography (ERG), MPS IIIB mice had a progressive decrease in the amplitude of the dark-adapted b-wave response. Corresponding pathology revealed shortening of the outer segments, thinning of the outer nuclear layer, and inclusions in the retinal pigmented epithelium. Auditory-evoked brainstem responses (ABR) demonstrated progressive hearing deficits consistent with the observed loss of hair cells in the inner ear and histologic abnormalities in the middle ear. The mouse model of MPS IIIB has several quantifiable phenotypic alterations and is similar to the human disease. These physiologic and histologic changes provide insights into the progression of this disease and will serve as important parameters when evaluating various therapies.

Carcinoma cells can acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT). However, the significance of EMT in cancer metastasis has been controversial, and the exact fates and functions of EMT cancer cells in vivo remain inadequately understood. Here, we tracked epithelial cancer cells that underwent inducible or spontaneous EMT in various tumor transplantation models. Unlike epithelial cells, the majority of EMT cancer cells were specifically located in the perivascular space and closely associated with blood vessels. EMT markedly activated multiple pericyte markers in carcinoma cells, in particular PDGFR-β and N-cadherin, which enabled EMT cells to be chemoattracted towards and physically interact with endothelium. In tumor xenografts generated from carcinoma cells that were prone to spontaneous EMT, a substantial fraction of the pericytes associated with tumor vasculature were derived from EMT cancer cells. Depletion of such EMT cells in transplanted tumors diminished pericyte coverage, impaired vascular integrity, and attenuated tumor growth. These findings suggest that EMT confers key pericyte attributes on cancer cells. The resulting EMT cells phenotypically and functionally resemble pericytes and are indispensable for vascular stabilization and sustained tumor growth. This study thus proposes a previously unrecognized role for EMT in cancer.

The two hyaluronan synthases (HASs) fromStreptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His₆ tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239–4245). Kinetic characterization of the enzymes in isolated membranes gave K _m values for UDP-GlcUA of 40 ± 4 μm for spHAS and 51 ± 5 μm for seHAS. In both cases, theV _max profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of ∼2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The K _mvalues for UDP-GlcNAc were 60 ± 7 μm for seHAS and 149 ± 3 μm for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8–9 days of storage at –80 °C without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their K _mvalues for both substrates and a decrease inV _max. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higherK _m values for both substrates than the membrane-bound enzymes. The K _UDP-GlcUA for purified spHAS and seHAS increased 2–4-fold. TheK _UDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher K _mvalues, the V _max values for the purified HASs were only ∼50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n _H ∼ 2), whereas purified seHAS showed no cooperativity.