Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNAi screen was used to identify drivers of vascular invasion by panning shRNA library transduced non-invasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the LATS1 (large tumor suppressor 1) kinase that inhibits the activity of the transcriptional coactivator YAP in the Hippo pathway. Depletion of LATS1 from non-invasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent up-regulation of the cytokines IL6, IL8, and CXCL1, 2, and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Fibroblast growth factors (FGFs) participate in embryonic development, in maintenance of tissue homeostasis in the adult, and in various diseases. FGF-binding proteins (FGFBP) are secreted proteins that chaperone FGFs stored in the extracellular matrix to their receptor, and can thus modulate FGF signaling. FGFBP1 (alias BP1, FGF-BP1, or HBp17) expression is required for embryonic survival, can modulate FGF-dependent vascular permeability in embryos, and is an angiogenic switch in human cancers. To determine the function of BP1 in vivo, we generated tetracycline-regulated conditional BP1 transgenic mice. BP1-expressing adult mice are viable, fertile, and phenotypically indistinguishable from their littermates. Induction of BP1 expression increased mouse primary fibroblast motility in vitro, increased angiogenic sprouting into subcutaneous matrigel plugs in animals and accelerated the healing of excisional skin wounds. FGF-receptor kinase inhibitors blocked these effects. Healing skin wounds showed increased macrophage invasion as well as cell proliferation after BP1 expression. Also, BP1 expression increased angiogenesis during the healing of skin wounds as well as after ischemic injury to hindlimb skeletal muscles. We conclude that BP1 can enhance FGF effects that are required for the healing and repair of injured tissues in adult animals. The family of fibroblast growth factors (FGFs) encompasses 18 distinct FGF receptor ligands, with a wide expression range and a significant role in angiogenesis, tumor progression, wound healing, and embryonic development.1-4 Many members of the FGF family, such as FGF1 and FGF2, are immobilized in the extracellular matrix (ECM) bound to heparan sulfate proteoglycans (HSPGs) and released from this storage site by proteases and heparanases. 4 -6 The involvement of carrier proteins that shuttle FGFs from their storage site to their receptors represents an alternative mode of regulation of FGF release from the ECM. 1,7 FGF-binding protein 1 (BP1, FGFBP1, FGF-BP1, or HBp17), 8 the best characterized of the three known secreted FGFBPs, 9 is an extracellular chaperone that binds FGF1, 2, 7, 10, and 22 in a reversible, noncovalent manner. 8,10 -12 Binding of the C-terminus of the BP1 protein is sufficient for its interaction with FGF2.13 After binding to BP1, the biochemical and biological activities of FGF are positively modulated.14 Several findings from different laboratories indicate that BP1 can contribute to embryonic development, 15 angiogenesis, tumor growth, and malignant progression, 11,12,14 -20 as well as the maintenance and reinnervation of the neuromuscular junction. 21 We reported earlier that expression of BP1 in SW13 cells induces FGF2 release from the cells, FGF-dependent colony formation in soft agar, and the growth of highly vascularized tumors in nude mice.11 In contrast, depletion of endogenous BP1 from ME180 cells was observed to reduce the release of ECM bound FGF2 into the cell supernatants and to increase FGF2 immobilized on the cell surface.16 Consisten...

The key molecular events required for the formation of Ductal Carcinoma in Situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here we show that the nuclear receptor coactivator Amplified In Breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal 3D mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both prior to DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24−/CD44+ Breast Cancer Initiating Cells (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. Loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, HER2 and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 plays a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through maintenance of BCIC, facilitates formation of DCIS, a necessary step prior to development of invasive disease.

Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression.

Metastatic spread of cancer cells from the primary tumor site to distant organs is the major cause of death in cancer patients. To disseminate, cancer cells detach from the primary tumor, enter the blood stream and extravasate at distant organ sites such as the liver, lung, bone or brain. While cancer cells are known to evade contact inhibition during growth in culture, we found that cell density is still sensed and can signal through the Hippo pathway effectors LATS1 and YAP. These effectors control cancer cell invasive behavior into stromal tissues, expression of cytokines that recruit inflammatory cells and progression toward metastatic spread. In this perspective, we discuss the drivers and the significance of pathways controlled by cell growth density.