Alternative splicing in EMT and TGF-β signaling during cancer progression.

Epithelial to mesenchymal transition (EMT) is a physiological process during development where epithelial cells transform to acquire mesenchymal characteristics, which allows them to migrate and colonize secondary tissues. Many cellular signaling pathways and master transcriptional factors exert a myriad of controls to fine tune this vital process to meet various developmental and physiological needs. Adding to the complexity of this network are post-transcriptional and post-translational regulations.

GPCR Screening Reveals that the Metabolite Receptor HCAR3 Regulates Epithelial Proliferation, Migration, and Cellular Respiration.

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and GPCRs play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation.

A GPCR screening in human keratinocytes identifies that the metabolite receptor HCAR3 controls epithelial proliferation, migration, and cellular respiration.

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and G protein-coupled receptors (GPCRs) play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation.

Smurf2 Regulates Inflammation and Collagen Processing in Cutaneous Wound Healing through Transforming Growth Factor-β/Smad3 Signaling.

Wound healing is a highly conserved process that restores the integrity and functionality of injured tissues. Transforming growth factor (TGF)-β is a master regulator of wound healing, whose signaling is attenuated by the E3 ubiquitin ligase Smurf2. Herein, the roles of Smurf2 in cutaneous wound healing were examined using a murine incisional cutaneous model.

Transforming Growth Factor-β: An Agent of Change in the Tumor Microenvironment.

Transforming Growth Factor-β (TGF-β) is a key regulator of embryonic development, adult tissue homeostasis, and lesion repair. In tumors, TGF-β is a potent inhibitor of early stage tumorigenesis and promotes late stage tumor progression and metastasis. Here, we review the roles of TGF-β as well as components of its signaling pathways in tumorigenesis.

Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells.

Chromosomal aneuploidy is a defining feature of epithelial cancers. The pattern of aneuploidies is cancer-type specific. For instance, the gain of chromosome 13 occurs almost exclusively in colorectal cancer.

TGF-β-induced alternative splicing of TAK1 promotes EMT and drug resistance.

Transforming growth factor-β (TGF-β) is major inducer of epithelial-to-mesenchymal transition (EMT), which associates with cancer cell metastasis and resistance to chemotherapy and targeted drugs, through both transcriptional and non-transcriptional mechanisms. We previously reported that, in cancer cells, heightened mitogenic signaling allows TGF-β-activated Smad3 to interact with poly(RC) binding protein 1 (PCBP1) and together they regulate many alternative splicing events that favors expression of protein isoforms essential for EMT, cytoskeletal rearrangement, and adherens junction signaling. Here we show that the exclusion of TGF-β-activated kinase 1 (TAK1) variable exon 12 requires another RNA-binding protein, Fox-1 homolog 2 (Rbfox2), which binds intronic sequences in front of exon 12 independently of the Smad3-PCBP1 complex.

Cell motility in cancer invasion and metastasis: insights from simple model organisms.

Metastasis remains the greatest challenge in the clinical management of cancer. Cell motility is a fundamental and ancient cellular behaviour that contributes to metastasis and is conserved in simple organisms. In this Review, we evaluate insights relevant to human cancer that are derived from the study of cell motility in non-mammalian model organisms.

Lysophosphatidic acid regulates the motility of MCF10CA1a breast cancer cell sheets via two opposing signaling pathways.

Aberrant cell migration leads to the dispersal of malignant cells. The ubiquitous lipid mediator lysophosphatidic acid (LPA) modulates cell migration and is implicated in tumor progression. Yet, the signaling cascades that regulate LPA's effect on cell motility remain unclear.

Using the Dot Assay to Analyze Migration of Cell Sheets.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites.

Moving in and Out: Dispersion of Cells in Self-Generated Gradients.

Migrating cells can influence the direction of their own migration by metabolizing chemoattractants present in their environment. This is illustrated by the dispersal of melanoma cells, which break down lysophosphatidic acid and generate a gradient with increasing concentrations of lysophosphatidic acid distant from the tumor. Melanoma cells can then disperse away from the tumor as they migrate in the self-generated lysophosphatidic acid gradient.

How Little Is Too Much? - How Transient Tumor-Stromal Crosstalk Can Control Tumor Progression.

Tumorigenesis is driven by genetic and physiological alterations of tumor cells as well as by the host microenvironment. In a co-culture of breast cancer cells and fibroblasts, short term interactions between tumor cells and stromal fibroblasts increase levels of active, fibroblast derived TGF-β in the extracellular medium, which in turn induces an expanded metastatic pattern of MCF10CA1a cells. These findings suggest that the effects of stromal TGF-β on tumor cell phenotype can be modeled as a dynamical system rather than a continuous linear system.

Improving the design of the agarose spot assay for eukaryotic cell chemotaxis.

Migration of cells along gradients of effector molecules, i.e., chemotaxis, is necessary in immune response and is involved in development and cancer metastasis.

SDF-1α mediates wound-promoted tumor growth in a syngeneic orthotopic mouse model of breast cancer.

Increased growth of residual tumors in the proximity of acute surgical wounds has been reported; however, the mechanisms of wound-promoted tumor growth remain unknown. Here, we used a syngeneic, orthotopic mouse model of breast cancer to study mechanisms of wound-promoted tumor growth. Our results demonstrate that exposure of metastatic mouse breast cancer cells (4T1) to SDF-1α, which is increased in wound fluid, results in increased tumor growth.

Real-time motion analysis reveals cell directionality as an indicator of breast cancer progression.

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics.

Biological responses to TGF-β in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression.

TGF-β plays a dual role in epithelial carcinogenesis with the potential to either suppress or promote tumor progression. We found that levels of Smad3 mRNA, a critical mediator of TGF-β signaling, are reduced by approximately 60% in human breast cancer. We therefore used conditionally immortalized mammary epithelial cells (IMEC) of differing Smad3 genotypes to quantitatively address the Smad3 requirement for different biologic responses to TGF-β.

Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo.

Introduction: Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland.

Introduction: Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference (RNAi)-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ/Smad pathway as an example.

Methods: We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse) which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes.

Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer.

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers.

Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern of Smad3 signaling.

Canonical TGF-beta is involved in cell differentiation, tissue maintenance, and wound healing, but also plays a central role in the pathogenesis of diseases such as cancer Here we describe a lentivirus-based reporter vector system expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) under the control of a Smad3-responsive element (CAGA)12 that allows observation of the temporospatial pattern of endogeneous Smad3-mediated signaling on a cellular level. Use of this method will be valuable to identify cells with active Smad3 signaling and investigate the role of endogenous Smad3 signaling in complex systems such as co-cultures in vitro, or in tumors during tumor cell invasion and metastasis in vivo.

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer.

Smad4-expression is decreased in breast cancer tissues: a retrospective study.

Background: Although transforming growth factor beta (TGF-beta) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-beta signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer.

Methods: Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998.