SMAD4 represses FOSL1 expression and pancreatic cancer metastatic colonization.

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-ß/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.

Epigenetic Reprogramming of Cancer-Associated Fibroblasts Deregulates Glucose Metabolism and Facilitates Progression of Breast Cancer.

The mechanistic contributions of cancer-associated fibroblasts (CAFs) in breast cancer progression remain to be fully understood. While altered glucose metabolism in CAFs could fuel cancer cells, how such metabolic reprogramming emerges and is sustained needs further investigation. Studying fibroblasts isolated from patients with benign breast tissues and breast cancer, in conjunction with multiple animal models, we demonstrate that CAFs exhibit a metabolic shift toward lactate and pyruvate production and fuel biosynthetic pathways of cancer cells. The depletion or suppression of the lactate production of CAFs alter the tumor metabolic profile and impede tumor growth. The glycolytic phenotype of the CAFs is in part sustained through epigenetic reprogramming of HIF-1α and glycolytic enzymes. Hypoxia induces epigenetic reprogramming of normal fibroblasts, resulting in a pro-glycolytic, CAF-like transcriptome. Our findings suggest that the glucose metabolism of CAFs evolves during tumor progression, and their breast cancer-promoting phenotype is partly mediated by oxygen-dependent epigenetic modifications.

Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis.

Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis.

Cancer cells can divert metabolites into anabolic pathways to support their rapid proliferation and to accumulate the cellular building blocks required for tumour growth. However, the specific bioenergetic profile of invasive and metastatic cancer cells is unknown. Here we report that migratory/invasive cancer cells specifically favour mitochondrial respiration and increased ATP production. Invasive cancer cells use the transcription coactivator peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A, also known as PGC-1α) to enhance oxidative phosphorylation, mitochondrial biogenesis and the oxygen consumption rate. Clinical analysis of human invasive breast cancers revealed a strong correlation between PGC-1α expression in invasive cancer cells and the formation of distant metastases. Silencing of PGC-1α in cancer cells suspended their invasive potential and attenuated metastasis without affecting proliferation, primary tumour growth or the epithelial-to-mesenchymal program. Inherent genetics of cancer cells can determine the transcriptome framework associated with invasion and metastasis, and mitochondrial biogenesis and respiration induced by PGC-1α are also essential for functional motility of cancer cells and metastasis.

Rab23 regulates Nodal signaling in vertebrate left-right patterning independently of the Hedgehog pathway.

Asymmetric fluid flow in the node and Nodal signaling in the left lateral plate mesoderm (LPM) drive left-right patterning of the mammalian body plan. However, the mechanisms linking fluid flow to asymmetric gene expression in the LPM remain unclear. Here we show that the small GTPase Rab23, known for its role in Hedgehog signaling, plays a separate role in Nodal signaling and left-right patterning in the mouse embryo. Rab23 is not required for initial symmetry breaking in the node, but it is required for expression of Nodal and Nodal target genes in the LPM. Microinjection of Nodal protein and transfection of Nodal cDNA in the embryo indicate that Rab23 is required for the production of functional Nodal signals, rather than the response to them. Using gain- and loss-of function approaches, we show that Rab23 plays a similar role in zebrafish, where it is required in the teleost equivalent of the mouse node, Kupffer׳s vesicle. Collectively, these data suggest that Rab23 is an essential component of the mechanism that transmits asymmetric patterning information from the node to the LPM.

Identification of human epididymis protein-4 as a fibroblast-derived mediator of fibrosis.

The functional contribution of myofibroblasts in fibrosis is not well understood. Using a new genetic mouse model to track and isolate myofibroblasts, we performed gene expression profiling followed by biological validation to identify HE4 (encoding human epididymis protein 4, also known as WAP 4-disulfide core domain-2 or Wfdc2) as the most upregulated gene in fibrosis-associated myofibroblasts. The HE4 gene encodes for a putative serine protease inhibitor that is upregulated in human and mouse fibrotic kidneys and is elevated in the serum of patients with kidney fibrosis. HE4 suppresses the activity of multiple proteases, including serine proteases and matrix metalloproteinases, and specifically inhibits their capacity to degrade type I collagen. In particular, we identified two serine proteases, Prss35 and Prss23, as HE4 targets with functional relevance in kidney fibrosis. Administration of HE4-neutralizing antibodies accelerated collagen I degradation and inhibited fibrosis in three different mouse models of renal disease. Collectively these studies suggest that HE4 is a potential biomarker of renal fibrosis and a new therapeutic target.

A Wnt-bmp feedback circuit controls intertissue signaling dynamics in tooth organogenesis.

Many vertebrate organs form through the sequential and reciprocal exchange of signaling molecules between juxtaposed epithelial and mesenchymal tissues. We undertook a systems biology approach that combined the generation and analysis of large-scale spatiotemporal gene expression data with mouse genetic experiments to gain insight into the mechanisms that control epithelial-mesenchymal signaling interactions in the developing mouse molar tooth. We showed that the shift in instructive signaling potential from dental epithelium to dental mesenchyme was accompanied by temporally coordinated genome-wide changes in gene expression in both compartments. To identify the mechanism responsible, we developed a probabilistic technique that integrates regulatory evidence from gene expression data and from the literature to reconstruct a gene regulatory network for the epithelial and mesenchymal compartments in early tooth development. By integrating these epithelial and mesenchymal gene regulatory networks through the action of diffusible extracellular signaling molecules, we identified a key epithelial-mesenchymal intertissue Wnt-Bmp (bone morphogenetic protein) feedback circuit. We then validated this circuit in vivo with compound genetic mutations in mice that disrupted this circuit. Moreover, mathematical modeling demonstrated that the structure of the circuit accounted for the observed reciprocal signaling dynamics. Thus, we have identified a critical signaling circuit that controls the coordinated genome-wide expression changes and reciprocal signaling molecule dynamics that occur in interacting epithelial and mesenchymal compartments during organogenesis.

Pericyte depletion results in hypoxia-associated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway.

The functional role of pericytes in cancer progression remains unknown. Clinical studies suggest that low numbers of vessel-associated pericytes correlated with a drop in overall survival of patients with invasive breast cancer. Using genetic mouse models or pharmacological inhibitors, pericyte depletion suppressed tumor growth but enhanced metastasis. Pericyte depletion was further associated with increased hypoxia, epithelial-to-mesenchymal transition (EMT), and Met receptor activation. Silencing of Twist or use of a Met inhibitor suppressed hypoxia and EMT/Met-driven metastasis. In addition, poor pericyte coverage coupled with high Met expression in cancer cells speculates the worst prognosis for patients with invasive breast cancer. Collectively, our study suggests that pericytes within the primary tumor microenvironment likely serve as important gatekeepers against cancer progression and metastasis.

VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization.

Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.