Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers.

The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2ß1 integrin, which is present only on basal and stem cells. The presence of ß1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.

Author Correction: Hysteresis control of epithelial-mesenchymal transition dynamics conveys a distinct program with enhanced metastatic ability.

The original version of this Article contained an error in the spelling of the author Daniel D. Liu, which was incorrectly given as Daniel Liu. This has now been corrected in both the PDF and HTML versions of the Article.

Hysteresis control of epithelial-mesenchymal transition dynamics conveys a distinct program with enhanced metastatic ability.

Epithelial-mesenchymal transition (EMT) have been extensively characterized in development and cancer, and its dynamics have been modeled as a non-linear process. However, less is known about how such dynamics may affect its biological impact. Here, we use mathematical modeling and experimental analysis of the TGF-ß-induced EMT to reveal a non-linear hysteretic response of E-cadherin repression tightly controlled by the strength of the miR-200s/ZEBs negative feedback loop. Hysteretic EMT conveys memory state, ensures rapid and robust cellular response and enables EMT to persist long after withdrawal of stimuli. Importantly, while both hysteretic and non-hysteretic EMT confer similar morphological changes and invasive potential of cancer cells, only hysteretic EMT enhances lung metastatic colonization efficiency. Cells that undergo hysteretic EMT differentially express subsets of stem cell and extracellular matrix related genes with significant clinical prognosis value. These findings illustrate distinct biological impact of EMT depending on the dynamics of the transition.

MicroRNAs as regulators of bone homeostasis and bone metastasis.

MicroRNAs (miRNAs) are short, endogenous RNAs that have essential roles in regulating gene expression through the disruption of target genes. The miRNA-induced suppression can occur through Argonaute-mediated cleavage of target mRNAs or by translational inhibition. System-wide studies have underscored the integral role that miRNAs play in regulating the expression of essential genes within bone marrow stromal cells. The miRNA expression has been shown to enhance or inhibit cell differentiation and activity, and elucidating miRNA targets within bone marrow cells has revealed novel regulations during normal bone development. Importantly, multiple studies have shown that miRNA misexpression mediates the progression of bone-related pathologies, including osteopetrosis and osteoporosis, as well as the development and progression of osteosarcoma. Furthermore, recent studies have detailed the capacity for miRNAs to influence bone metastasis from a number of primary carcinomas. Taken together, these findings reveal the significant clinical potential for miRNAs to regulate bone homeostasis, as well as to mediate bone-related pathologies.

The microRNA-23b/27b/24 cluster promotes breast cancer lung metastasis by targeting metastasis-suppressive gene prosaposin.

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.

Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis.

Understanding the mechanism by which tumor cells influence osteoclast differentiation is crucial for improving treatment of osteolytic metastasis. Here, we report broad microRNA (miRNA) expression changes in differentiating osteoclasts after exposure to tumor-conditioned media, in part through activation of NFκB signaling by soluble intracellular adhesion molecule (sICAM1) secreted from bone-metastatic cancer cells. Ectopic expression of multiple miRNAs downregulated during osteoclastogenesis suppresses osteoclast differentiation by targeting important osteoclast genes. Intravenous delivery of these miRNAs in vivo inhibits osteoclast activity and reduces osteolytic bone metastasis. Importantly, serum levels of sICAM1 and two osteoclast miRNAs, miR-16 and miR-378, which are elevated in osteoclast differentiation, correlate with bone metastasis burden. These findings establish miRNAs as potential therapeutic targets and clinical biomarkers of bone metastasis.

Transcriptional control of cancer metastasis.

Transcriptional regulation is an essential component of tumor progression and metastasis. During cancer progression, dysregulation of oncogenic or tumor-suppressive transcription factors (TFs), as well as master cell fate regulators and tumor microenvironment-induced factors, collectively influence multiple steps of the metastasis cascade, including local invasion, dissemination, and eventual colonization of the tumor to distant organs. Furthermore, epigenetic alterations in tumor cells, including DNA methylation, as well as activation or suppression of histone deacetylases (HDACs), histone acetyltransferases (HATs), and other chromatin-modifying enzymes, can further distort the transcriptional network to influence metastasis. We focus here on recent research advances in transcriptional control of metastasis and highlight the therapeutic potential of targeting such transcriptional regulatory networks.

Vascular endothelial-cadherin stimulates syndecan-1-coupled insulin-like growth factor-1 receptor and cross-talk between αVß3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis.

Vascular endothelial growth factor (VEGF)-stimulated angiogenesis depends on a cross-talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)-cadherin and the αVß3 integrin. Because we have shown that αVß3 integrin activation is dependent on its incorporation, along with the insulin-like growth factor-1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan-1 (Sdc1), we questioned the role of this core complex in VEGF-stimulated angiogenesis. We find that the Sdc1-coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE-cadherin) engagement. VE-cadherin binding to Fc/VE-cadherin extracellular domain chimera activates Sdc1-coupled IGF1R and αvß3 integrin; this depends on VEGFR2 and c-Src activated by the cadherin. Blocking homotypic VE-cadherin engagement disrupts VEGF-stimulated cell migration, which is restored by clustering the cadherin in the absence of cell-cell adhesion. This cadherin-dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)(92-119), a peptide that competitively disrupts the Sdc1-coupled ternary complex and prevents the αVß3 integrin activation required for VEGFR2 activation. VEGFR2-stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVß3 integrin comprises a core activation mechanism activated by VE-cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.

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.

Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor.

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the alpha(v)beta(3) and alpha(v)beta(5) integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1's interaction with these integrins. Because the alpha(v)beta(3) and alpha(v)beta(5) integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.