Dingcheng Gao

Endothelial Progenitor Cells Control the Angiogenic Switch in Mouse Lung Metastasis

Angiogenesis-mediated progression of micrometastasis to lethal macrometastasis is the major cause of death in cancer patients. Here, using mouse models of pulmonary metastasis, we identify bone marrow (BM)–derived endothelial progenitor cells (EPCs) as critical regulators of this angiogenic switch. We show that tumors induce expression of the transcription factor Id1 in the EPCs and that suppression of Id1 after metastatic colonization blocked EPC mobilization, caused angiogenesis inhibition, impaired pulmonary macrometastases, and increased survival of tumor-bearing animals. These findings establish the role of EPCs in metastatic progression in preclinical models and suggest that selective targeting of EPCs may merit investigation as a therapy for cancer patients with lung metastases.

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.The role of epithelial to mesenchymal transition (EMT) in metastasis is a longstanding source of controversy, largely due to an inability to monitor transient and reversible EMT phenotypes in vivo. We established an EMT lineage tracing system to monitor this process, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We confirmed that within a predominantly epithelial primary tumor, a small portion of tumor cells undergo EMT. Strikingly, lung metastases mainly consisted of non-EMT tumor cells maintaining their epithelial phenotype. Inhibiting EMT by overexpressing miR-200 did not impact lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Microenvironmental Regulation of Epithelial–Mesenchymal Transitions in Cancer

The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. Activation of the epithelial-to-mesenchymal transition (EMT) endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs. The observation that in many cancers distant metastases resemble the epithelial phenotype of primary tumors has led to speculation that the disseminated tumor cells recruited to the target organs undergo mesenchymal-to-epithelial transition (MET). However, the MET cascade has not been recapitulated in vivo, and the cellular and molecular regulators that promote MET remain unknown. In a recent report, using a model of spontaneous breast cancer, we have shown that bone marrow-derived myeloid progenitor cells in the premetastatic lung secrete the proteoglycan versican, which induces MET of metastatic tumor cells and accelerates metastases. This review summarizes recent progress in MET research, outlines a unique paracrine cross-talk between the microenvironment and the cancer cells, which promotes tumor outgrowth in the metastatic organ, and discusses opportunities for novel antimetastatic approaches for cancer therapy.

Myeloid Progenitor Cells in the Premetastatic Lung Promote Metastases by Inducing Mesenchymal to Epithelial Transition

Tumors systemically initiate metastatic niches in distant target metastatic organs. These niches, composed of bone marrow-derived hematopoietic cells, provide permissive conditions for future metastases. However, the mechanisms by which these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhanced recruitment of bone marrow-derived CD11b(+)Gr1(+) myeloid progenitor cells in the premetastatic lungs. Gene expression profiling revealed that the myeloid cells from metastatic lungs express versican, an extracellular matrix proteoglycan. Notably, versican in metastatic lungs was mainly contributed by the CD11b(+)Ly6C(high) monocytic fraction of the myeloid cells and not the tumor cells or other stromal cells. Versican knockdown in the bone marrow significantly impaired lung metastases in vivo, without impacting their recruitment to the lungs or altering the immune microenvironment. Versican stimulated mesenchymal to epithelial transition of metastatic tumor cells by attenuating phospho-Smad2 levels, which resulted in elevated cell proliferation and accelerated metastases. Analysis of clinical specimens showed elevated versican expression within the metastatic lung of patients with breast cancer. Together, our findings suggest that selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease.

Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression

Emerging evidence indicates that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated growth of certain tumors in mice and human. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. While the contributions of EPCs to neovessel formation in spontaneous and transplanted tumors and to the metastatic transition have been reported to be relatively low, remarkably, specific EPC ablation in vivo has resulted in severe angiogenesis inhibition and impaired primary and metastatic tumor growth. The existence of a BM reservoir of EPCs, and the selective involvement of EPCs in neovascularization, have attracted considerable interest because these cells represent novel target for therapeutic intervention. In addition, EPCs are also being used as pharmacodynamic surrogate markers for monitoring cancer progression, as well as for optimizing efficacy of anti-angiogenic therapies in the clinic. This review will focus primarily on recent advances and emerging concepts in the field of EPC biology and discuss ongoing debates involving the role of EPCs in tumor neovascularization. For detailed information on the in vitro characterization of EPCs contribution to non-tumor pathologies, the reader is directed towards several excellent reviews and publications [F. Bertolini, Y. Shaked, P. Mancuso and R.S. Kerbel, Nat. Rev., Cancer 6 (2006) 835-845. [1]] [J.M. Hill, T. Finkel and A.A. Quyyumi, Vox Sang. 87 Suppl 2 (2004) 31-37. [2]] [A.Y. Khakoo and T. Finkel, Annu. Rev. Med. 56 (2005) 79-101. [3]] [H.G. Kopp, C.A. Ramos and S. Rafii, Curr. Opin. Hematol. 13 (2006) 175-181. [4]; K.K. Hirschi, D.A. Ingram and M.C. Yoder, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584-1595. [5]; F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove and J. Case, J. Cell. Mol. Med. 13 (2009) 87-102. [6]] and reviews by Bertolini, Voest and Yoder in this issue.

MicroRNAs regulate tumor angiogenesis modulated by endothelial progenitor cells

Bone marrow-derived endothelial progenitor cells (EPC) contribute to the angiogenesis-dependent growth of tumors in mice and humans. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. miRNAs have emerged as key regulators of several cellular processes including angiogenesis; however, whether miRNAs contribute to bone marrow-mediated angiogenesis has remained unknown. Here, we show that genetic ablation of miRNA-processing enzyme Dicer, specifically in the bone marrow, decreased the number of circulating EPCs, resulting in angiogenesis suppression and impaired tumor growth. Furthermore, genome-wide deep sequencing of small RNAs revealed tumor EPC-intrinsic miRNAs including miR-10b and miR-196b, which have been previously identified as key regulators of HOX signaling and adult stem cell differentiation. Notably, we found that both miR-10b and miR-196b are responsive to vascular endothelial growth factor stimulation and show elevated expression in human high-grade breast tumor vasculature. Strikingly, targeting miR-10b and miR-196b led to significant defects in angiogenesis-mediated tumor growth in mice. Targeting these miRNAs may constitute a novel strategy for inhibiting tumor angiogenesis.

Suppression of miRNA-708 by Polycomb Group Promotes Metastases by Calcium-Induced Cell Migration

The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.

The role of bone-marrow-derived cells in tumor growth, metastasis initiation and progression

Emerging evidence from murine models suggests that tumor-specific endocrine factors systemically stimulate the quiescent bone marrow (BM) compartment, resulting in the expansion, mobilization and recruitment of BM progenitor cells. Discrete subsets of tumor-instigated BM-derived progenitor cells support tumor progression and metastasis by regulating angiogenesis, inflammation and immune suppression. Notably, clinical studies have begun to reveal that increased BM recruitment in tumors is associated with poor prognosis. Thus, the BM-derived tumor microenvironment is an attractive therapeutic target, and drugs targeting the components of the microenvironment are currently in clinical trials. Here, we focus on recent advances and emerging concepts regarding the intriguing role of BM-derived cells in tumor growth, metastasis initiation and progression, and we discuss future directions in the context of novel diagnostic and therapeutic opportunities.

CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2

The versatility of Ca2+ as a second messenger lies in the complex manner in which Ca2+ signals are generated. How information contained within the Ca2+ code is interpreted underlies cell function. Recently, we identified CAPRI and RASAL as related Ca2+-triggered Ras GTPase-activating proteins. RASAL tracks agonist-stimulated Ca2+ oscillations by repetitively associating with the plasma membrane, yet CAPRI displays a long-lasting Ca2+-triggered translocation that is refractory to cytosolic Ca2+ oscillations. CAPRI behavior is Ca2+- and C2 domain–dependent but sustained recruitment is predominantly Ca2+ independent, necessitating integration of Ca2+ by the C2 domains with agonist-evoked plasma membrane interaction sites for the pleckstrin homology domain. Using an assay to monitor Ras activity in real time, we correlate the spatial and temporal translocation of CAPRI with the deactivation of H-Ras. CAPRI seems to low-pass filter the Ca2+ signal, converting different intensities of stimulation into different durations of Ras activity in contrast to the preservation of Ca2+ frequency information by RASAL, suggesting sophisticated modes of Ca2+-regulated Ras deactivation.