Publications

The thyroid hormone (T3) receptor type alpha, the c-ErbA alpha proto-oncoprotein, stimulates transcription of T3-dependent promoters, interferes with AP-1 activity, and induces erythroid differentiation in a ligand-dependent manner. The v-ErbA oncoprotein does not bind hormone and has lost all of these activities. Using c-ErbA/v-ErbA chimeras, we found that a deletion of 9 amino acids, conserved among many members of the nuclear receptor superfamily, which are located at the extreme carboxy terminus of c-ErbA alpha is responsible for loss of both transactivation and transcriptional interference activities. Single, double, and triple amino acid substitutions within this region completely abolished T3-dependent transcriptional activation, interference with AP-1 activity, and decreased T3 binding by c-ErbA alpha. However, the lower T3 binding by these mutants does not fully account for the loss of transactivation and transcriptional interference, since a c-ErbA/v-ErbA chimera which was similarly reduced in T3 binding activity has retained both of these functions. Deletion of homologous residues in the retinoic acid receptor alpha (RAR alpha) resulted in a similar loss of transactivation and transcriptional interference activities. The ability of c-ErbA alpha to induce differentiation of transformed erythroblasts is also impaired by all of the mutations introduced into the conserved carboxy-terminal sequence. We conclude that this 9-amino-acid conserved region is essential for normal biological function of c-ErbA alpha and RAR alpha and possibly other T3 and RA receptors.

585 The purpose of this study was to validate the laboratory generated equation that predicted speed, and indirectly METS, from vector magnitude (VM) counts generated by the Tritrac, in a field setting. Thirty non-obese young adults were instructed, to "stroll", "walk briskly" and "jog at a comfortable pace" for two laps each around a flat, outdoor, cork 400m track. Exercise bouts were timed and actual speeds calculated. Projected speeds calculated using field generated VM data and the laboratory developed regression equation that predicted speed from VM counts were found to be significantly different from actual speeds (p < .05). Actual speeds were underestimated by 0.69, 0.53, 0.94, and 0.73 mph for the light, moderate, vigorous, and total activity. Correlation demonstrated a strong linear relationship between pooled actual speeds and VM data (R2e = .90, p<.01). The results of a two-way repeated measures ANOVA indicated a significant speed effect (p=.001), a significant gender effect (p=.019), and a significant interaction between speed and gender (p=.001). Women ran significantly slower than men during the vigorous activity but VM did not underestimate the speed as it did in men. Using the laboratory developed VM cut-points for light, moderate, and vigorous activity, 33% of the light and 20% of the moderate activity bouts were incorrectly classified. These results may indicate that laboratory developed VM cut-points established to classify activity may provide misleading results in a field setting. Possible differences in the biomechanics of treadmill versus overland walking and running may explain the results. A new regression equation that estimated overground walking and running speeds from VM data was developed (speed = .001175*VM + .474). Supported by NIH 49505

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

Abstract Obesity is a risk factor for the development of insulin resistance/type 2 diabetes and several types of cancer, including cancers of the colon, breast, liver, endometrium and esophagus. Obese individuals have increased levels of free fatty acids (FFAs) in their serum, and so we are investigating signaling pathways that are activated by FFAs in vitro to gain insight into pathways that may trigger insulin resistance and the activation of oncogenes in obese individuals in vivo. Obesity is associated with chronic activation of c-Jun NH2-terminal kinase (JNK)1 in liver, fat and muscle tissue, and studies using JNK1-deficient mice demonstrate that JNK activation is important for adiposity, peripheral insulin resistance and the production of the proinflammatory cytokines, but the mechanism of JNK1 activation in obesity is unclear. Our data demonstrate that JNK1 is activated by FFAs in vitro in a c-Src-dependent manner, and that activation of c-Src occurs within lipid rafts. Src-deficient murine embryonic fibroblasts (MEFs) and SYF MEFs, which are deficient in c-Src family kinases c-Src, c-Yes and c-Fyn, do not activate JNK1 after exposure to FFAs in vitro, while NIH 3T3 MEFs and SYF MEFs reconstituted with c-Src activate JNK1 normally. In contrast, SYF MEFs reconstituted with c-Yes and c-Fyn do not activate JNK1. Knockdown of c-Src with shRNA in HEK 293T cells also inhibits FFA-induced activation of JNK1, and inhibition of c-Src with pharmacologic inhibitors has the same effect in J774 mouse macrophages. Pharmacologic inhibition of c-Src also prevents the FFA-induced upregulation of tumor necrosis factor (TNF)-α mRNA in macrophages. Importantly, c-Src is activated within lipid rafts after FFA treatment, as seen by both detergent-dependent and detergent-independent methods of raft isolation. Interestingly, omega-3 polyunsaturated fatty acids (PUFAs), which can protect against the onset of obesity, will also block JNK activation following treatment with saturated FFAs, and we suspect that this effect may be due to the ability of PUFAs to exclude c-Src from lipid rafts. Finally, preliminary data suggests that c-Src may be aberrantly activated in liver tissue from obese mice that were fed a high-fat diet. Experiments are currently underway to verify the role of c-src in the activation of JNK1 in vivo by acutely infusing mice with a free fatty acid emulsion in the presence of a pharmacologic inhibitor of c-src. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 38.

SUMMARY Current approaches to stage chronic liver diseases have limited utility to directly predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and chronically injured pre-malignant livers using two distinct mouse models. Analysis of 40,748 hepatic nuclei unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers, but were increasingly prevalent as chronic liver disease progressed towards hepatocarcinogenesis. Gene expression deconvolution of 1,439 human liver transcriptomes from publicly available datasets revealed that daHep frequencies highly correlate with current histopathological liver disease staging systems. Importantly, we show that high daHep levels precede carcinogenesis in mice and humans and predict a higher risk of hepatocellular carcinoma (HCC) development. This novel transcriptional signature with diagnostic and, more importantly, prognostic significance has the potential to change the way chronic liver disease patients are staged, surveilled and risk-stratified.

An online version of the Microscale Audit of Pedestrian Streetscapes (Abbreviated) tool was adapted to virtually audit built environment features supportive of physical activity. The current study assessed inter-rater reliability of MAPS Online between in-person raters and online raters unfamiliar with the regions. In-person and online audits were conducted for a total of 120 quarter-mile routes (60 per site) in Phoenix, AZ and San Diego, CA. Routes in each city included 40 residential origins stratified by walkability and SES, and 20 commercial centers. In-person audits were conducted by raters residing in their region. Online audits were conducted by raters in the alternate location using Google Maps (Aerial and Street View) images. The MAPS Abbreviated Online tool consisted of four sections: overall route, street segments, crossings and cul-de-sacs. Items within each section were grouped into subscales, and inter-rater reliability (ICCs) was assessed for subscales at multiple levels of aggregation. Online and in-person audits showed excellent agreement for overall positive microscale (ICC = 0.86, 95% CI [0.80, 0.90]) and grand scores (ICC = 0.93, 95% CI [0.89, 0.95]). Substantial to near-perfect agreement was found for 21 of 30 (70%) subscales, valence, and subsection scores, with ICCs ranging from 0.62, 95% CI [0.50, 0.72] to 0.95, 95% CI [0.93, 0.97]. Lowest agreement was found for the aesthetics and social characteristics scores, with ICCs ranging from 0.07, 95% CI [−0.12, 0.24] to 0.27, 95% CI [0.10, 0.43]. Results support use of the MAPS Abbreviated Online tool to reliably assess microscale neighborhood features that support physical activity and may be used by raters residing in different geographic regions and unfamiliar with the audit areas.

Although epithelial NF-κB signaling is important for lung carcinogenesis, NF-κB inhibitors are ineffective for cancer treatment. To explain this paradox, we studied mice with genetic deletion of IKKβ in myeloid cells and found enhanced tumorigenesis in Kras(G12D) and urethane models of lung cancer. Myeloid-specific inhibition of NF-κB augmented pro-IL-1β processing by cathepsin G in neutrophils, leading to increased IL-1β and enhanced epithelial cell proliferation. Combined treatment with bortezomib, a proteasome inhibitor that blocks NF-κB activation, and IL-1 receptor antagonist reduced tumor formation and growth in vivo. In lung cancer patients, plasma IL-1β levels correlated with poor prognosis, and IL-1β increased following bortezomib treatment. Together, our studies elucidate an important role for neutrophils and IL-1β in lung carcinogenesis and resistance to NF-κB inhibitors.

The tumor microenvironment (TME) provides potential targets for cancer therapy. However, how signals originating in cancer cells affect tumor-directed immunity is largely unknown. Deletions in the <i>CHUK</i> locus, coding for IκB kinase α (IKKα), correlate with reduced lung adenocarcinoma (ADC) patient survival and promote Kras^G12D-initiated ADC development in mice, but it is unknown how reduced IKKα expression affects the TME. Here, we report that low IKKα expression in human and mouse lung ADC cells correlates with increased monocyte-derived macrophage and regulatory T cell (Treg) scores and elevated transcription of genes coding for macrophage-recruiting and Treg-inducing cytokines (CSF1, CCL22, TNF, and IL-23A). By stimulating recruitment of monocyte-derived macrophages from the bone marrow and enforcing a TNF/TNFR2/c-Rel signaling cascade that stimulates Treg generation, these cytokines promote lung ADC progression. Depletion of TNFR2, c-Rel, or TNF in CD4⁺ T cells or monocyte-derived macrophages dampens Treg generation and lung tumorigenesis. Treg depletion also attenuates carcinogenesis. In conclusion, reduced cancer cell IKKα activity enhances formation of a protumorigenic TME through a pathway whose constituents may serve as therapeutic targets for KRAS-initiated lung ADC.