Erik H. J. Danen

Integrins: Signaling, disease, and therapy

Background: Integrins are a family of transmembrane receptors that mediate cell-cell and cell-matrix adhesion. They are involved in stable cell adhesion and migration of cells. In addition, integrin-mediated interactions modulate the response to most, if not all growth factors, cytokines, and other soluble factors. Purpose: In this review, we briefly explain how integrins can affect the multitude of signal transduction cascades in control of survival, proliferation, and differentiation. Subsequently, we primarily focus on targeting integrins α5β1 and αvβ3 in disease and we discuss how antagonists of these integrins, including disintegrins, RGD peptides, small molecules, and function blocking antibodies, may be of therapeutical value either alone or, especially in the treatment of cancer, in combination with existing therapeutical strategies.

Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time‐resolved imaging of both processes at single‐cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two‐photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

Drug‐induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF‐α). HepG2 cells were treated with diclofenac followed by TNF‐α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF‐α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF‐α‐mediated enhancement involved activation of caspase‐3 through a caspase‐8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c‐Jun N‐terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF‐α‐induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF‐α‐mediated nuclear factor kappaB (NF‐κB) translocation oscillation in association with reduced NF‐κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF‐α‐induced phosphorylation of the inhibitor of NF‐κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS‐345541 as well as stable lentiviral short hairpin RNA (shRNA)‐based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF‐α‐induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac‐mediated stress signaling suppresses TNF‐α‐induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;)

Automated whole animal bio-imaging assay for human cancer dissemination

A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

β1 Integrin Inhibition Elicits a Prometastatic Switch Through the TGFβ–miR-200–ZEB Network in E-Cadherin–Positive Triple-Negative Breast Cancer

Although some breast cancer therapies reduce the primary tumor, they may trigger unwanted metastasis. When Treatment Promotes Metastasis In cancer, therapy aims to kill the primary tumor and prevent metastasis. Truong et al. found that although strategies blocking β1 integrin are effective at treating primary breast tumors, they may cause metastatic disease in certain patients. In E-cadherin–positive triple-negative breast cancer (TNBC) cell lines, blocking β1 integrin function by gene silencing or with antibodies induced epithelial-to-mesenchymal transition (EMT)–associated signaling and the loss of E-cadherin, enabling TNBC cells to migrate individually and invade a three-dimensional collagen matrix in culture. When injected into zebrafish, the β1 integrin–deficient cells disseminated further than the parent TNBC cells. When implanted in mice, these cells formed more lung metastases, despite producing smaller primary tumors compared with those produced by implanted parent cells. Thus, therapies targeted against β1 integrin may not be suitable for some TNBC patients. Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the β1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of β1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked β1 integrin function or knockdown of β1 switched the migratory behavior of human and mouse E-cadherin–positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor–β (TGFβ) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box–binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFβ receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in β1 integrin–deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that β1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting β1 integrins may have undesirable effects in TNBC.

The cancer stem cell microenvironment and anti-cancer therapy

Purpose: Tumours are composed of a heterogeneous cell population. Cancer stem cells, which make up a minor fraction of a tumour, may be the cells that initiate and sustain tumour growth. Cancer stem cells are believed to share many properties with normal stem cells that render them relatively insensitive to classical radio- and chemotherapy. Conclusions: We discuss what those (cancer) stem cell properties are and how the interactions with the microenvironment – ‘the niche’ – control those aspects of (cancer) stem cell biology. We also describe possible strategies to target cancer stem cells in order to prevent cancers from escaping therapy.

Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing, or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins -- the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.

Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

ABSTRACT Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response.

The integrin expression profile modulates orientation and dynamics of force transmission at cell–matrix adhesions

ABSTRACT Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins &agr;5&bgr;1 and &agr;v&bgr;3, changes, thus altering the integrin composition of cell–matrix adhesions. Here, we show that enhanced &agr;v&bgr;3 expression can fully compensate for loss of &agr;5&bgr;1 and other &bgr;1 integrins to support outside-in and inside-out force transmission. &agr;5&bgr;1 and &agr;v&bgr;3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, &agr;5&bgr;1 and &agr;v&bgr;3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using &agr;v&bgr;3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by &agr;5&bgr;1. Thus, altering the relative abundance of fibronectin-binding integrins in cell–matrix adhesions affects the spatiotemporal organization of force transmission.

A guide to mechanobiology: Where biology and physics meet.

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates. This article is part of a Special Issue entitled: Mechanobiology.