Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling

Abstract

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.

Cell Density Regulates Cancer Metastasis Via the Hippo Pathway

“…We observed that cancer cells grown at low density present a highly invasiveness phenotype through upregulation of several cytokines including IL8, CXCL1, 2 and 3 as YAP appear to translocate to the nucleus. Inhibition of YAP-TEAD association using the inhibitor Verteporfin reversed this upregulation [7]. These findings matched with the YAP-dependent control of inflammatory cytokine expression and signaling in the mouse model of pancreatic cancer driven by mutant KRAS mentioned above [24].…”

“…The less compacted cells with nuclear YAP localization, give rise to the trophoectoderm while the more compacted cells in the inner cell mass show cytoplasmic localization of YAP [15,[30][31]. This is somewhat reminiscent of observations during malignant progression [7]: YAP cellular localization regulates the invasive capability of cancer cells and cancer cells forming lung metastases at the endothelial boundary of blood vessels showed nuclear YAP staining in contrast to cells distant from the endothelial invasion zone (Figure 2). Quite strikingly, signaling molecules involved in organ size control mechanisms coordinated by YAP also appear to contribute to invasive cancers [32][33][34].…”

“…We have found that the density at which cells are grown in tissue culture regulates the vascular invasiveness of frequently studied cancer cell lines. Cells grown at high density were less successful at invading an endothelial monolayer in vitro and at metastasis in vivo than cells grown at low density [7]. Quite strikingly, several known aggressive cancer cell lines showed highly malleable invasive behavior in response to altered cell density in culture (summarized in Table 1).…”

“…Our group has shown the contribution of the Hippo pathway to cancer cell/ endothelial crosstalk and vascular invasion. Quite surprisingly, some of the most aggressive cancer [7].…”

“…In mechanistic studies with cultured cells and animal models, cytokines were associated with a more invasive cancer cell phenotype. Also, cancer cells that express CXCL1 Adapted from [7,24]. www.futuremedicine.com and 2 attract mesenchymal cells in response to the cytokines and render the cancers more metastatic and resilient to chemotherapy [36].…”