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Objective Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor β (TGFβ) signaling or through distinct signal transduction pathways. Methods We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen α2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. Results Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibro-blasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFβ. Conclusion These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.

BackgroundBreast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naïve and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers.MethodsPatient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing.ResultsForty-eight breast cancers (24 post-chemotherapy, 24 chemo-naïve) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p<0.001). Genomic losses and gains could be detected in the BCX, and three models demonstrated a transformation to induce mouse tumors. However, overall, somatic mutation profiles including potential drivers were maintained upon implantation and serial passaging. One BCX model was cultured in vitro and re-implanted, maintaining its genomic profile.ConclusionsBCXs can be established from clinically aggressive breast cancers, especially in TNBC patients with poor response to NeoCT. Future studies will determine the potential of in vivo models for identification of genotype-phenotype correlations and individualization of treatment.

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations-widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery-justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

The mammalian target of rapamycin (mTOR) is a central controller of cell growth and is currently being investigated as a potential target in breast cancer therapy. The essential amino acid leucine has been proposed to regulate mTOR signaling. The objective of this study was to determine whether leucine restriction would inhibit mTOR signaling in breast cancer cells. Leucine restriction did not decrease mTOR signaling in any of the eight breast cancer cell lines tested. In addition, in vivo administration of a leucine-free diet for up to four days did not result in a decrease in phosphorylation of mTOR target proteins in breast cancer xenografts. Further, in three different cell lines, an increase in Akt phosphorylation was observed after leucine restriction. This was observed without a decrease in S6K phosphorylation, suggesting a mechanism different from the feedback loop activation of Akt observed with rapamycin treatment. We conclude that leucine restriction is not sufficient to inhibit mTOR signaling in most breast cancer cell lines, but is associated with activation of survival molecule Akt, making leucine deprivation an undesirable approach for breast cancer therapy.