Yibin Kang

A multigenic program mediating breast cancer metastasis to bone

We investigated the molecular basis for osteolytic bone metastasis by selecting human breast cancer cell line subpopulations with elevated metastatic activity and functionally validating genes that are overexpressed in these cells. These genes act cooperatively to cause osteolytic metastasis, and most of them encode secreted and cell surface proteins. Two of these genes, interleukin-11 and CTGF, encode osteolytic and angiogenic factors whose expression is further increased by the prometastatic cytokine TGF beta. Overexpression of this bone metastasis gene set is superimposed on a poor-prognosis gene expression signature already present in the parental breast cancer population, suggesting that metastasis requires a set of functions beyond those underlying the emergence of the primary tumor.

The miR-200 Family Inhibits Epithelial-Mesenchymal Transition and Cancer Cell Migration by Direct Targeting of E-cadherin Transcriptional Repressors ZEB1 and ZEB2

MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-β-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.

Epithelial-Mesenchymal Transitions: Twist in Development and Metastasis

Epithelial-mesenchymal transitions (EMT) are vital for morphogenesis during embryonic development and are also implicated in the conversion of early stage tumors into invasive malignancies. Several key inducers of EMT are transcription factors that repress E-cadherin expression. A recent report in Cell (Yang et al., 2004) adds Twist to this list and links EMT to the ability of breast cancer cells to enter the circulation and seed metastases.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

MinireviewEpithelial-Mesenchymal Transitions: Twist in Development and Metastasis

Epithelial-mesenchymal transitions (EMT) are vital for morphogenesis during embryonic development and are also implicated in the conversion of early stage tumors into invasive malignancies. Several key inducers of EMT are transcription factors that repress E-cadherin expression. A recent report in Cell (Yang et al., 2004) adds Twist to this list and links EMT to the ability of breast cancer cells to enter the circulation and seed metastases.

A Self-Enabling TGFβ Response Coupled to Stress Signaling: Smad Engages Stress Response Factor ATF3 for Id1 Repression in Epithelial Cells

Genome-wide transcriptional profiling of human epithelial cells revealed that repression of Id inhibitors of differentiation (Id1, Id2, and Id3) is a general feature of the TGFbeta cytostatic program. Opposite responses of Id1 to TGFbeta and the related factor BMP are dictated by the specific ability of the TGFbeta mediator, Smad3, to activate expression of stress response factor ATF3 and then recruit this factor to the Id1 promoter. Thus, a Smad3-mediated primary gene response, ATF3 induction, enables Smad3 to participate in an ATF3-mediated, secondary gene response. As a common target of TGFbeta/Smad signals and stress signals via p38 kinase, ATF3 additionally serves to channel synergy between these pathways in the response of epithelial cells to stress and injury.

Beyond tumorigenesis: cancer stem cells in metastasis

The importance of cancer stem cells (CSCs) in tumor-initiation has been firmly established in leukemia and recently reported for a variety of solid tumors. However, the role of CSCs in multistage cancer progression, particularly with respect to metastasis, has not been well-defined. Cancer metastasis requires the seeding and successful colonization of specialized CSCs at distant organs. The biology of normal stem cells and CSCs share remarkable similarities and may have important implications when applied to the study of cancer metastasis. Furthermore, overlapping sets of molecules and pathways have recently been identified to regulate both stem cell migration and cancer metastasis. These molecules constitute a complex network of cellular interactions that facilitate both the initiation of the pre-metastasis niche by the primary tumor and the formation of a nurturing organ microenvironment for migrating CSCs. In this review, we surveyed the recent advances in this dynamic field and propose a unified model of cancer progression in which CSCs assume a central role in both tumorigenesis and metastasis. Better understanding of CSCs as a fundamental component of the metastatic cascade will lead to novel therapeutic strategies against metastatic cancer.

E2F4/5 and p107 as Smad Cofactors Linking the TGFβ Receptor to c-myc Repression

Smad3 is a direct mediator of transcriptional activation by the TGFbeta receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. We defined how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners.

Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization

Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin–dependent epithelial traits and Sec23a-mediated tumor cell secretome.

Hypoxia and hypoxia-inducible factors: master regulators of metastasis.

Hypoxia is a common condition found in a wide range of solid tumors and is often associated with poor prognosis. Hypoxia increases tumor glycolysis, angiogenesis, and other survival responses, as well as invasion and metastasis by activating relevant gene expressions through hypoxia-inducible factors (HIF). HIF-1α and HIF-2α undergo oxygen-dependent regulation, and their overexpression is frequently associated with metastasis and poor clinical outcomes. Recent studies show that each step of the metastasis process, from the initial epithelial-mesenchymal transition to the ultimate organotropic colonization, can potentially be regulated by hypoxia, suggesting a master regulator role of hypoxia and HIFs in metastasis. Furthermore, modulation of cancer stem cell self-renewal by HIFs may also contribute to the hypoxia-regulated metastasis program. The hypoxia-induced metastatic phenotype may be one of the reasons for the modest efficacy of antiangiogenic therapies and may well explain the recent provocative findings that antiangiogenic therapy increased metastasis in preclinical models. Multiple approaches to targeting hypoxia and HIFs, including HIF inhibitors, hypoxia-activated bioreductive prodrugs, and gene therapies may become effective treatments to prevent or reduce metastasis. Clin Cancer Res; 16(24); 5928–35 ©2010 AACR.