Angiogenesis : from tumor initiation to therapeutic resistance

Cancer is a leading cause of morbidity and mortality worldwide. In 2012 approximately 14 million new cases were diagnosed and 8.2 million cancer-related deaths were recorded. A better understanding of the strategies employed by cancer cells to grow and disseminate through the body is still required. Precise characterization of the signaling pathways involved in these processes will allow us to propose new diagnostic and prognostic markers but also to improve therapeutic strategies. Angiogenesis, the formation of new blood vessels from a pre-existing vasculature, has been proposed as a suitable target in order to curtail cancer. In particular, it has been proposed that preventing the supply of nutrients and oxygen supply to the tumor would starve it to death. However, the clinical outcome of anti-angiogenic therapy has been sobering; despite initial therapeutic effects, patients relapse with cancers that have developed resistance to the therapy. Tumors treated with bevacizumab, a monoclonal antibody targeting the master regulator of angiogenesis, Vascular Endothelial Growth Factor-A (VEGF-A), have been found to activate alternative pro-angiogenic signaling pathways in order to revascularize and resume growth. Therefore, it becomes critical to decipher the molecular mechanisms implicated in tumor angiogenesis in general but also the mechanisms underlying the development of resistance to anti-angiogenic therapies. In my Ph.D. thesis, I first aimed to decipher the mechanisms of resistance to anti-angiogenic therapy. In order to overcome revascularization through activation of alternative pro-angiogenic signaling pathways, several pan-tyrosine kinase inhibitors have been developed. They demonstrated increased efficacy compared with bevacizumab. Here, we assessed the efficacy of nintedanib, a multikinase inhibitor targeting VEGFRs, FGFRs and PDGFRs in a mouse model of breast cancer. While tumors primarily responded to nintedanib treatment and demonstrated decreased tumor mass after short-term treatment, prolonged nintedanib treatment was associated with tumor regrowth. However, angiogenesis was still repressed in tumors escaping therapy and no revascularization was observed. Microarray analysis of FAC-sorted tumor cells revealed a metabolic shift towards anaerobic glycolysis. Moreover, tumors established metabolic symbiosis as suggested by the alternation between highly hypoxic, glycolytic and normoxic areas. Indeed, the inhibition of glycolysis or the disruption of metabolic symbiosis by genetically ablating MCT4 expression, a protein involved in metabolic symbiosis, efficiently overcame resistance to anti-angiogenic therapy. In order to reach blood vessels and to metastasize, epithelial cancer cells have to gain motile properties. The first step of the metastatic cascade consists of an epithelial-mesenchymal transtition (EMT). Epithelial cells undergoing this program lose apico-basal polarity and their epithelial markers and cell-cell and cell-matrix contacts, yet express mesenchymal markers and gain migratory capacity. Moreover, cells undergoing an EMT acquire cancer stem cell (CSC) traits. Mesenchymal cells are, for example, able to initiate tumor formation in a more efficient way compared to epithelial cells. While this feature is expected to rely on increased self-renewal capacity in mesenchymal cells, our laboratory identified VEGF-A as a causal agent in tumor initiation. By secreting VEGF-A, mesenchymal cells induce a precocious angiogenic switch, therefore sustaining tumor growth. In a second project, I aimed to identify the upstream regulator of VEGF-A in cells undergoing an EMT. Here, by performing a low throughput siRNA screen for transcription factors possessing a binding site on the VEGF-A gene promoter, I could identify JunB as the main regulator of VEGF-A expression in mesenchymal cells. JunB inhibition in diverse mesenchymal cell lines led to decreased VEGF-A expression, suggesting a key role for JunB in EMT-induced angiogenesis and thus tumor growth. In summary, my Ph.D. work provided new insights into tumor angiogenesis as: - I identified a new mechanism of resistance to anti-angiogenic therapy, in which tumor cells resumed growth despite lack of blood vessels by switching their metabolism towards glycolysis. This work highlighted the use of glycolysis inhibitors to overcome anti-angiogenic resistance; - I highlighted a new role for JunB as a regulator of EMT-induced angiogenesis and tumor growth

An immature B cell population from peripheral blood serves as surrogate marker for monitoring tumor angiogenesis and anti-angiogenic therapy in mouse models

Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45dimVEGFR1⁻CD31low, that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45dimVEGFR1⁻CD31low cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45dimVEGFR1⁻CD31low cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer

Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

Despite the approval of several anti-angiogenic therapies, clinical results remain unsatisfactory, and transient benefits are followed by rapid tumor recurrence. Here, we demonstrate potent anti-angiogenic efficacy of the multi-kinase inhibitors nintedanib and sunitinib in a mouse model of breast cancer. However, after an initial regression, tumors resume growth in the absence of active tumor angiogenesis. Gene expression profiling of tumor cells reveals metabolic reprogramming toward anaerobic glycolysis. Indeed, combinatorial treatment with a glycolysis inhibitor (3PO) efficiently inhibits tumor growth. Moreover, tumors establish metabolic symbiosis, illustrated by the differential expression of MCT1 and MCT4, monocarboxylate transporters active in lactate exchange in glycolytic tumors. Accordingly, genetic ablation of MCT4 expression overcomes adaptive resistance against anti-angiogenic therapy. Hence, targeting metabolic symbiosis may be an attractive avenue to avoid resistance development to anti-angiogenic therapy in patients

An immature B cell population from peripheral blood serves as surrogate marker for monitoring tumor angiogenesis and anti-angiogenic therapy in mouse models

Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45dimVEGFR1−CD31low, that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45dimVEGFR1−CD31low cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45dimVEGFR1−CD31low cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer

Repression of malignant tumor progression upon pharmacological IGF-1R blockade in a mouse model of insulinoma

NVP-AEW541, a specific inhibitor of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase, has been reported to interfere with tumor growth in various tumor transplantation models. We have assessed the efficacy of NVP-AEW541 in repressing tumor growth and tumor progression in the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis. In addition, we have tested NVP-AEW541 in Rip1Tag2;RipIGF-1R double-transgenic mice which show accelerated tumor growth and increased tumor malignancy compared to Rip1Tag2 single-transgenic mice. Previously, we have shown that high levels of IGF-II, a high-affinity ligand for IGF-1R, are required for Rip1Tag2 tumor cell survival and tumor growth. Unexpectedly, treatment of Rip1Tag2 mice with NVP-AEW541 in prevention and intervention trials did neither affect tumor growth nor tumor cell proliferation and apoptosis. Yet, it significantly repressed progression to tumor malignancy, i.e. the rate of the transition from differentiated adenoma to invasive carcinoma. Treatment of Rip1Tag2;RipIGF1R double-transgenic mice resulted in moderately reduced tumor volumes and increased rates of tumor cell apoptosis. Sustained expression of IGF-II and of the IGF-II-binding form of insulin receptor (IR-A) in tumor cells suggests a compensatory role of IR-A upon IGF-1R blockade. The results indicate that inhibition of IGF-1R alone is not sufficient to efficiently block insulinoma growth and imply an overlapping role of IGF-1R and insulin receptor in executing mitogenic and survival stimuli elicited by IGF-II. The reduction of tumor invasion upon IGF-1R blockade on the other hand indicates a critical function of IGF-1R signaling for the acquisition of a malignant phenotype

VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation

An epithelial-mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A-mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation

Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinoma

Background: InformationTumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. Results: By employing mouse models expressing high and low levels of the laminin 1 chain (LM1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LM1, VEGFA expression triggered by the complex integrin 21-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. Conclusions: Together, we have identified VEGFA, CXCR4 and 21 integrin downstream of LM1 in colon cancer as of bad prognostic value for patient survival. Significance: This information opens novel opportunities for diagnosis and treatment of colon cancer