Faculty Bibliography

Tumor metastases and epithelial to mesenchymal transition (EMT) involve tumor cell invasion and migration through the dense collagen-rich extracellular matrix surrounding the tumor. Little is neither known about the mechanobiological mechanisms involved in this process, nor the role of the mechanical forces generated by the cells in their effort to invade and migrate through the stroma. In this paper we propose a new fundamental mechanobiological mechanism involved in cancer growth and metastasis, which can be both protective or destructive depending on the magnitude of the forces generated by the cells. This new mechanobiological mechanism directly challenges current paradigms that are focused mainly on biological and biochemical mechanisms associated with tumor metastasis. Our new mechanobiological mechanism describes how tumor expansion generates mechanical forces within the stroma to not only resist tumor expansion but also inhibit or enhance tumor invasion by, respectively, inhibiting or enhancing matrix metalloproteinase (MMP) degradation of the tensed interstitial collagen. While this mechanobiological mechanism has not been previously applied to the study of tumor metastasis and EMT, it may have the potential to broaden our understanding of the tumor invasive process and assist in developing new strategies for preventing or treating cancer metastasis.

Tumor cells undergoing the epithelial-mesenchymal transition (EMT) acquire the capacity to migrate, invade the stroma and metastasize. EMT cells also acquire stem cell characteristics suggesting crosstalk between EMT and stem cell pathways and contribution of the EMT process to the generation of cancer stem cells. Indeed, transforming growth factor-beta (TGF-β), a major inducer of EMT, cooperates with stem cell pathways like Wnt, Ras, Hedgehog and Notch to induce EMT. A molecular basis for this cooperative signaling is indicated by recent data showing that many EMT associated transcription factors like Snail1, Zeb1/2, Twist, β-catenin, Lef/TCF, Foxc2 and AP-1 interact with Smads and form EMT promoting Smad complexes (EPSC) engaged in both repressing epithelial genes and activating mesenchymal genes. Thus, formation and activation of EPSC seems to represent a point of convergence between EMT and stem cell pathways. Here, we review our current understanding of the mechanisms involved in the transcriptional crosstalk between TGF-β and stem cell pathways and discuss how a fundament for the activation of these mechanisms may lead to the induction of EMT in tumors.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT

Several oncogenic proteins and tumour suppressors target the RNA polymerase I and interfere with rRNA synthesis. Here, we show that the glycogen synthase kinase (GSK) 3beta, which phosphorylates the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), is selectively enriched in nucleoli of RAS-transformed cells. Immunoprecipitation and chromatin immunoprecipitation assays performed on epithelial and endothelial cells transformed with oncogenic RAS show that GSK3beta and PTEN are part of the same complex and associate with promoter and coding region of the rDNA. An active GSK3beta mutant abolished nucleolar BrUTP incorporation and associated with the member of the selectivity factor 1 complex TAF(I)110. Finally, GSK3beta inhibition upregulated 45S, 18S and 28S rRNA synthesis in RAS-transformed epithelial cells as revealed by semiquantitative real-time PCR and promoted cellular proliferation. Our results underscore a repressive function for GSK3beta in rRNA biogenesis supporting its role as a tumour supressor.

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

Endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% +/- 4% or 1% +/- 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% +/- 12% or 5% +/- 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% +/- 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.

Neutralizing antiviral antibodies are typically detected on the basis of inhibition of viral function, such as propagation of a viral infection or inhibition of viral gene expression. Evidence is presented that anti-adenovirus neutralizing antibodies can be evaluated by analysis of cell-associated capsids or by analysis of intracellular trafficking of the capsids within 1 h after infection. Quantitative analyses of these morphologic parameters represent rapid, broadly applicable, functional assays for the detection of anti-adenovirus neutralizing antibodies.

Fcgamma receptors convey to phagocytic cells the ability to recognize, bind, and internalize IgG-coated cells and microorganisms. The present study demonstrates the use of adenovirus (Ad)-mediated gene transfer of human Fcgamma receptor IIA cDNA to convert normally nonphagocytic cells (hepatocytes) into functional equivalents of phagocytic cells. Ad vector in vitro transfer and expression of FcgammaRIIA cDNA in primary rat hepatocytes was confirmed by flow cytometry anti-FcgammaRIIA immunodetection, and the function of the receptor was demonstrated by enhanced binding and phagocytosis of (51)Cr-labeled IgG-opsonized erythrocytes. After in vivo gene transfer to rats, expression of FcgammaRIIA cDNA in hepatocytes was confirmed by Northern analysis and immunohistochemistry. Rats infected with the Ad vector carrying the FcgammaRIIA cDNA demonstrated enhanced clearance of opsonized erythrocytes, but not nonopsonized erythrocytes, from the circulation with increased sequestration within the liver. Together, these data demonstrate that Ad-mediated FcgammaRIIA gene transfer can convert normally IgG-nonphagocytic cells into phagocytic cells capable of recognizing, binding, and ingesting an opsonized particulate antigen, suggesting that gene transfer strategies might be used to transiently augment host defense by enhancing the clearance of immune complexes.