Curzio Rüegg

The tumor microenvironment and its contribution to tumor evolution toward metastasis.

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.

Vascular Integrins in Tumor Angiogenesis: Mediators and Therapeutic Targets

The notion that tumor angiogenesis may have therapeutic implications in the control of tumor growth was introduced by Dr. Judah Folkman in 1971. The approval of Avastin in 2004 as the first antiangiogenic systemic drug to treat cancer patients came as a validation of this visionary concept and opened new perspectives to the treatment of cancer. In addition, this success boosted the field to the quest for new therapeutic targets and antiangiogenic drugs. Preclinical and clinical evidence indicate that vascular integrins may be valid therapeutic targets. In preclinical studies, pharmacological inhibition of integrin function efficiently suppressed angiogenesis and inhibited tumor progression. alphaVbeta3 and alphaVbeta5 were the first vascular integrins targeted to suppress tumor angiogenesis. Subsequent experiments revealed that at least four additional integrins (i.e., alpha1beta1, alpha2beta1, alpha5beta1, and alpha6beta4) might be potential therapeutic targets. In clinical studies low-molecular-weight integrin inhibitors and anti-integrin function-blocking antibodies demonstrated low toxicity and good tolerability and are now being tested in combination with radiotherapy and chemotherapy for anticancer activity in patients. In this article the authors review the role of integrins in angiogenesis, present recent development in the use of alphaVbeta3 and alpha5beta1 integrin antagonists as potential therapeutics in cancer, and discuss future perspectives.

Integrin Inhibitors Reaching the Clinic

Malignant cellular transformation is largely viewed as the consequence of autonomous cell genetic alterations, leading to the activation of oncogenes and the inactivation of tumor suppression genes and increasing genomic instability, which gives rise to a cell capable of limitless proliferation and survival, invasion, and metastasis. In addition, the formation of a tumor-associated vasculature—a process also referred to as tumor angiogenesis—has been recognized as an essential event promoting tumor progression. In the absence of tumor angiogenesis, tumors enter a state of dormancy, characterized by a balance between cell proliferation and apoptosis. During the last decade, significant advances have been made in the understanding of the tissue, cellular, and molecular events that regulate and mediate tumor angiogenesis. Many extracellular, cell surface, and intracellular molecules modulating angiogenesis have been identified and characterized, including growth factors and growth factor receptors, such as vascular endothelial growth factor (VEGF) and VEGF receptors; integrin and cadherin adhesion molecules; remodeling and guidance molecules and their receptors (eg, ephrin/Eph, angiopoietins/tyrosine kinase with Ig and EGF homology domains [Tie-2]); matrix-degrading enzymes (eg, matrix metalloproteinase [MMP] -9), signaling molecules, and transcription factors (eg, hypoxia inducible factor 1 alpha [HIF1 ]). Many of these molecules, in particular VEGF, have been or are being evaluated as novel therapeutic targets. Most recently, vascular integrin inhibition is being tested as antiangiogenic therapy. Integrins are heterodimer transmembrane receptors for the extracellular matrix composed of an alpha and beta subunit. Natural integrin ligands include laminin, fibronectin, and vitronectin, but they also include fibrinogen and fibrin, thrombospondin, MMP-2, and fibroblast growth factor 2. Integrins bind ligands by recognizing short amino acid stretches on exposed loops, particularly the arginine-glycine-aspartic acid (RGD) sequence. On ligation, integrins mediate complex signaling events, alone or in combination with growth factor receptors, regulating cell adhesion, proliferation, survival, and migration by activating canonical pathways, such as integrin-linked kinase (ILK), protein kinase B (PKB/Akt), mitogen-activated protein kinase (MAPK), Rac or nuclear factor kappa B (NFB). In resting vessels, integrins interact with the basal membrane, thereby maintaining vascular quiescence. During angiogenesis, they are essential for endothelial cell migration, proliferation, and survival. In preclinical studies, pharmacologic inhibition of integrin function efficiently suppressed angiogenesis and inhibited tumor progression. Of the 24 known integrin heterodimers, V 3 and V 5 were the first vascular integrins targeted to suppress tumor angiogenesis. These encouraging preclinical results stimulated researchers and industry to develop pharmacologic inhibitors of integrin function for clinical testing. Three classes of integrin inhibitors are currently in preclinical and clinical development: monoclonal antibodies targeting the extracellular domain of the heterodimer (eg, Vitaxin; MedImmune, Gaithersburg, MD), synthetic peptides containing an RGD sequence (eg, cilengitide; Merck KGaA, Darmstadt, Germany), and peptidomimetics (eg, S247; Pfizer, St Louis, MO), which are orally bioavailable nonpeptidic molecules mimicking the RGD sequence. In this issue of the Journal of Clinical Oncology, Nabors et al, on behalf of the New Approaches to Brain Tumor Therapy consortium, report on a phase I trial of cilengitide conducted in patients with recurrent glioma. Cilengitide (EMD 121974) is a cyclic RGD-motif containing peptide binding with high specificity to the V 3 and V 5 receptors. Glioblastoma, a highly vascularized and invasive tumor with microvascular proliferation as one of its hallmarks, is a logical target for such therapy. Overexpression of V 3 and V 5 has been shown in a majority of glioblastoma. In this trial, cilengitide was administered intravenously twice weekly at increasing doses up to 2,400 mg/m, without reaching dose-limiting toxicity. Overall tolerance was excellent, with occasional patients experiencing joint and bone pain. Thrombosis, thrombocytopenia, electrolyte imbalance, and anorexia were also reported. In five of the 51 patients, an objective response was observed. Sixteen other patients are reported as stable for a median duration of 5 months (range, 3 to 11 months). The pharmacokinetic profile was as reported previously, with no influence of comedications, in particular the frequent administration of enzymeinducing antiepileptic drugs. These results are intriguing for several reasons. First, despite a wide range of dosages, no clear pattern of toxicity could be determined. The absence of bleeding episodes is reassuring, as hemorrhage might have been expected with such a therapy and in this tumor type in particular. Second, objective and even long-lasting responses were seen, although mechanistically one would not necessarily expect measurable tumor reduction, but rather slowing of the growth curve. Due to the inherent heterogeneity of the patient and tumor population in phase I trials, the growth rate cannot be assessed in this trial. The authors refrain from reporting the progression-free survival rate at 3 or 6 months, which are commonly used surrogate end points in brain tumor trials. Third, responses were seen both at the lower dose levels (360 mg/m) as well as at the highest dose level tested (2,400 mg/m), allowing no conclusion to what dose is the most appropriate to be evaluated in future studies. Last, the authors are to be commended for having attempted to include correlative imaging end points in this study. However, imaging end points require careful standardization and a complex statistical analysis. Their data suggest that the JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 13 MAY 1 2007

Low Doses of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Myeloid cells contribute to tumor lymphangiogenesis.

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation.

Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation‐dependent signaling pathways

Summary.  Background: Recent evidence indicates that zoledronate, a nitrogen‐containing bisphosphonate used to treat conditions of increased bone resorption, may have anti‐angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. Objectives: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. Results: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3‐kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. Conclusions: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation‐dependent manner.

N-cadherin as a therapeutic target in cancer

During tumor progression, cancer cells undergo dramatic changes in the expression profile of adhesion molecules resulting in detachment from original tissue and acquisition of a highly motile and invasive phenotype. A hallmark of this change, also referred to as the epithelial–mesenchymal transition, is the loss of E- (epithelial) cadherin and the de novo expression of N- (neural) cadherin adhesion molecules. N-cadherin promotes tumor cell survival, migration and invasion, and a high level of its expression is often associated with poor prognosis. N-cadherin is also expressed in endothelial cells and plays an essential role in the maturation and stabilization of normal vessels and tumor-associated angiogenic vessels. Increasing experimental evidence suggests that N-cadherin is a potential therapeutic target in cancer. A peptidic N-cadherin antagonist (ADH-1) has been developed and has entered clinical testing. In this review, the authors discuss the biochemical and functional features of N-cadherin, its potential role in cancer and angiogenesis, and summarize the preclinical and clinical results achieved with ADH-1.

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC‐MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids‐containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.

Metabolic Labeling and Protein Linearization Technology Allow the Study of Proteins Secreted by Cultured Cells in Serum-Containing Media

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Fc Block Treatment, Dead Cells Exclusion, and Cell Aggregates Discrimination Concur to Prevent Phenotypical Artifacts in the Analysis of Subpopulations of Tumor-Infiltrating CD11b(+) Myelomonocytic Cells

It is well established that cancer cells can recruit CD11b+ myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor‐infiltrating CD11b+ myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564–1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b+ myeloid cells isolated from peripheral blood and from tumors, using six‐color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor‐derived samples concur to generate artifacts in the phenotype of tumor‐infiltrating CD11b+ subpopulations. We show that the heterogeneity of tumor‐infiltrating CD11b+ subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor‐infiltrating CD11b+ cells was particularly sensitive to these parameters compared to circulating CD11b+ cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor‐infiltrating CD11b+ cell populations.