Progression of breast cancer to metastatic bone disease is linked to deregulated expression of the transcription factor Runx2. Therefore, our goal was to evaluate the potential for clinical use of Runx2-targeting microRNAs (miRNAs) to reduce tumor growth and bone metastatic burden. Expression analysis of a panel of miRNAs regulating Runx2 revealed a reciprocal relationship between the abundance of Runx2 protein and two miRNAs, miR-135 and miR-203. These miRNAs are highly expressed in normal breast epithelial cells where Runx2 is not detected, and absent in metastatic breast cancer cells and tissue biopsies that express Runx2. Reconstituting metastatic MDA-MB-231-Luc cells with miR-135 and miR-203 reduced the abundance of Runx2 and expression of the metastasis-promoting Runx2 target genes IL-11, MMP-13, and PTHrP. Additionally, tumor cell viability was decreased and migration suppressed in vitro. Orthotopic implantation of MDA-MB-231-luc cells delivered with
miR-135 or miR-203, followed by an intratumoral administration of the synthetic miRNAs reduced the tumor growth and spontaneous metastasis to bone. Furthermore, intratibial injection of these miRNA-delivered cells impaired tumor growth in the bone environment and inhibited bone resorption. Importantly, reconstitution of Runx2 in MDA-MB-231-luc cells delivered with miR-135 and miR-203 reversed the inhibitory effect of the miRNAs on tumor growth and metastasis. Thus, we have identified that aberrant expression of Runx2 in aggressive tumor cells is related to the loss of specific Runx2-targeting miRNAs and that a clinically relevant replacement strategy by delivery of synthetic miRNAs is a candidate therapeutic approach to prevent metastatic bone disease by this route.

Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFβ stimulation. Although some TECs strikingly upregulate α smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFβ treatment. Compared with NECs, SMA+ TECs were 40% less motile in wound-healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreenCdh5-Cre reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA+ TECs, suggesting that not all endothelial cells (EC) respond identically to TGFβ in vivo. Indeed, examination of 84 TGFβ-regulated target genes revealed entirely different genetic signatures in TGFβ-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFβ-regulated genes but operates in tandem with TGFβ to upregulate others. ECs challenged with TGFβ secrete bFGF, which blocks SMA expression in secondary cultures, suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFβ-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature. Cancer Res; 75(7); 1–11. ©2015 AACR.

Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFβ stimulation. Whereas some TEC strikingly up-regulate alpha smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFβ treatment. Compared with NEC, SMA+ TEC were 40% less motile in wound healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreenCdh5-Cre reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA+ TEC, suggesting that not all endothelial cells (EC) respond identically to TGFβ in vivo. Indeed, examination of 84 TGFβ-regulated target genes revealed entirely different genetic signatures in TGFβ-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFβ-regulated genes but operates in tandem with TGFβ to up-regulate others. EC challenged with TGFβ secrete bFGF which blocks SMA expression in secondary cultures suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFβ-driven EndMT produces a spectrum of endothelial cell phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature.

Tumor-initiating cells (TIC) perpetuate tumor growth, enable therapeutic resistance, and drive initiation of successive tumors. Virtually nothing is known about the role of mechanotransductive signaling in controlling TIC tumorigenesis, despite the recognized importance of altered mechanics in tissue dysplasia and the common observation that extracellular matrix (ECM) stiffness strongly regulates cell behavior. To address this open question, we cultured primary human glioblastoma (GBM) TICs on laminin-functionalized ECMs spanning a range of stiffnesses. Surprisingly, we found that these cells were largely insensitive to ECM stiffness cues, evading the inhibition of spreading, migration, and proliferation typically imposed by compliant ECMs. We hypothesized that this insensitivity may result from insufficient generation of myosin-dependent contractile force. Indeed, we found that both pharmacologic and genetic activation of cell contractility through RhoA GTPase, Rho-associated kinase, or myosin light chain kinase restored stiffness-dependent spreading and motility, with TICs adopting the expected rounded and nonmotile phenotype on soft ECMs. Moreover, constitutive activation of RhoA restricted three-dimensional invasion in both spheroid implantation and Transwell paradigms. Orthotopic xenotransplantation studies revealed that control TICs formed tumors with classical GBM histopathology including diffuse infiltration and secondary foci, whereas TICs expressing a constitutively active mutant of RhoA produced circumscribed masses and yielded a 30% enhancement in mean survival time. This is the first direct evidence that manipulation of mechanotransductive signaling can alter the tumor-initiating capacity of GBM TICs, supporting further exploration of these signals as potential therapeutic targets and predictors of tumor-initiating capacity within heterogeneous tumor cell populations. Cancer Res; 75(6); 1–10. ©2014 AACR.

Tumor-initiating cells (TICs) perpetuate tumor growth, enable therapeutic resistance, and drive initiation of successive tumors. Virtually nothing is known about the role of mechanotransductive signaling in controlling TIC tumorigenesis, despite the recognized importance of altered mechanics in tissue dysplasia and the common observation that extracellular matrix (ECM) stiffness strongly regulates cell behavior. To address this open question, we cultured primary human glioblastoma (GBM) TICs on laminin-functionalized ECMs spanning a range of stiffnesses. Surprisingly, we found that these cells were largely insensitive to ECM stiffness cues, evading the inhibition of spreading, migration, and proliferation typically imposed by compliant ECMs. We hypothesize that this insensitivity may result from insufficient generation of myosin-dependent contractile force. Indeed, we found that both pharmacologic and genetic activation of cell contractility through RhoA GTPase, Rho-associated kinase (ROCK), or myosin light chain kinase (MLCK) restored stiffness-dependent spreading and motility, with TICs adopting the expected rounded and non-motile phenotype on soft ECMs. Moreover, constitutive activation of RhoA restricted three-dimensional invasion in both spheroid implantation and transwell paradigms. Orthotopic xenotransplantation studies revealed that control TICs formed tumors with classical GBM histopathology including diffuse infiltration and secondary foci, whereas TICs expressing a constitutively active mutant of RhoA produced circumscribed masses and yielded a 30% enhancement in mean survival time. This is the first direct evidence that manipulation of mechanotransductive signaling can alter the tumor-initiating capacity of GBM TICs, supporting further exploration of these signals as potential therapeutic targets and predictors of tumor initiating capacity within heterogeneous tumor cell populations.