Alessia Bottos

Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways

BackgroundOsteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory.ResultsWe investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice.ConclusionIn conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.

The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions

Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain. We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against β-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of β-neurexin, we show that the anti-β-neurexin antibody influences vessel tone in isolated chicken arteries. Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.

Targeting oncogenic serine/threonine-protein kinase BRAF in cancer cells inhibits angiogenesis and abrogates hypoxia

Carcinomas are comprised of transformed epithelial cells that are supported in their growth by a dedicated neovasculature. How the genetic milieu of the epithelial compartment influences tumor angiogenesis is largely unexplored. Drugs targeted to mutant cancer genes may act not only on tumor cells but also, directly or indirectly, on the surrounding stroma. We investigated the role of the BRAFV600E oncogene in tumor/vessel crosstalk and analyzed the effect of the BRAF inhibitor PLX4720 on tumor angiogenesis. Knock-in of the BRAFV600E allele into the genome of human epithelial cells triggered their angiogenic response. In cancer cells harboring oncogenic BRAF, the inhibitor PLX4720 switches off the ERK pathway and inhibits the expression of proangiogenic molecules. In tumor xenografts harboring the BRAFV600E, PLX4720 extensively modifies the vascular network causing abrogation of hypoxia. Overall, our results provide a functional link between oncogenic BRAF and angiogenesis. Furthermore, they indicate how the tumor vasculature can be “indirectly” besieged through targeting of a genetic lesion to which the cancer cells are addicted.

Decreased NK-cell tumour immunosurveillance consequent to JAK inhibition enhances metastasis in breast cancer models

The JAK/STAT pathway is an attractive target for breast cancer therapy due to its frequent activation, and clinical trials evaluating JAK inhibitors (JAKi) in advanced breast cancer are ongoing. Using patient biopsies and preclinical models of breast cancer, we demonstrate that the JAK/STAT pathway is active in metastasis. Unexpectedly, blocking the pathway with JAKi enhances the metastatic burden in experimental and orthotopic models of breast cancer metastasis. We demonstrate that this prometastatic effect is due to the immunosuppressive activity of JAKi with ensuing impairment of NK-cell-mediated anti-tumour immunity. Furthermore, we show that immunostimulation with IL-15 overcomes the enhancing effect of JAKi on metastasis formation. Our findings highlight the importance of evaluating the effect of targeted therapy on the tumour environment. The impact of JAKi on NK cells and the potential value of immunostimulators to overcome the weakened tumour immunosurveillance, are worthwhile considering in the clinical setting of breast cancer.

Oncogenes and angiogenesis: a way to personalize anti-angiogenic therapy?

The acquisition of oncogenic mutations and promotion of angiogenesis are key hallmarks of cancer. These features are often thought of as separate events in tumor progression and the two fields of research have frequently been considered as independent. However, as we highlight in this review, activated oncogenes and deregulated angiogenesis are tightly associated, as mutations in cancer cells can lead to perturbation of the pro- and anti-angiogenic balance thereby causing aberrant angiogenesis. We propose that normalization of the vascular network by targeting oncogenes in the tumor cells might lead to more efficient and sustained therapeutic effects compared to therapies targeting tumor vessels. We discuss how pharmacological inhibition of oncogenes in tumor cells restores a functional vasculature by bystander anti-angiogenic effect. As genetic alterations are tumor-specific, targeted therapy, which potentially blocks the angiogenic program activated by individual oncogenes may lead to personalized anti-angiogenic therapy.

Transcriptional regulation of tenascin-W by TGF-beta signaling in the bone metastatic niche of breast cancer cells.

Tenascin‐W is a matricellular protein with a dynamically changing expression pattern in development and disease. In adults, tenascin‐W is mostly restricted to stem cell niches, and is also expressed in the stroma of solid cancers. Here, we analyzed its expression in the bone microenvironment of breast cancer metastasis. Osteoblasts were isolated from tumor‐free or tumor‐bearing bones of mice injected with MDA‐MB231‐1833 breast cancer cells. We found a fourfold upregulation of tenascin‐W in the osteoblast population of tumor‐bearing mice compared to healthy mice, indicating that tenascin‐W is supplied by the bone metastatic niche. Transwell and co‐culture studies showed that human bone marrow stromal cells (BMSCs) express tenascin‐W protein after exposure to factors secreted by MDA‐MB231‐1833 breast cancer cells. To study tenascin‐W gene regulation, we identified and analyzed the tenascin‐W promoter as well as three evolutionary conserved regions in the first intron. 5′RACE analysis of mRNA from human breast cancer, glioblastoma and bone tissue showed a single tenascin‐W transcript with a transcription start site at a noncoding first exon followed by exon 2 containing the ATG translation start. Site‐directed mutagenesis of a SMAD4‐binding element in proximity of the TATA box strongly impaired promoter activity. TGFβ1 induced tenascin‐W expression in human BMSCs through activation of the TGFβ1 receptor ALK5, while glucocorticoids were inhibitory. Our experiments show that tenascin‐W acts as a niche component for breast cancer metastasis to bone by supporting cell migration and cell proliferation of the cancer cells.

Neuroligin 1 Induces Blood Vessel Maturation by Cooperating with the α6 Integrin

Background: Disregulated adhesion of endothelial cells to extracellular matrix proteins is associated with numerous human diseases. Results: Neuroligin 1 modulates the interaction between α6 integrin and laminin and induces the formation of a normal extracellular matrix. Conclusion: Neuroligin 1 modulates vessel maturation and cell adhesion. Significance: We first report that Neuroligin 1, originally a synaptic protein, modulates extracellular matrix functions in the vascular system. The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.

Cancer: Staying together on the road to metastasis

Most deaths from breast cancer occur when the primary tumour spreads to secondary sites. It now emerges that clusters of tumour cells that enter the bloodstream form metastases more often than single circulating tumour cells.

Memo has a novel role in S1P signaling and crucial for vascular development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Abstract LB-013: The immunomodulatory effect of JAK inhibitors enhances metastasis by impairing antitumor immunity in preclinical models of breast cancer

The JAK-STAT pathway is an attractive therapeutic target in breast cancer due to its frequent activation. Clinical trials testing JAK inhibitors in breast cancer are ongoing, making it important to understand the effect of this therapeutic approach on metastasis. While it is recognized that tumor growth in primary and metastatic sites is influenced by the local environment, little is known about the effect of targeted therapies on metastases or on host cells interacting with tumor cells at distant locations. Our goal was to determine the effect of JAK inhibitors on breast cancer in the bone environment, a common site of metastasis. Using patient biopsies and preclinical models of breast cancer metastasis, we demonstrate that the JAK-STAT pathway is active in bone metastasis, both in the cancer cells and in the tumor environment. To study the effect of JAK inhibitors, we established preclinical models of bone metastatic tumors, taking advantage of the bone tropism when injected via the intracardiac route, of the breast cancer cell lines, human MDA-MB231 scp1833 and mouse EO771. In vivo, in both models, STAT3 was active and treatment with the JAK1-JAK2 inhibitor ruxolitinib decreased pSTAT3 levels in primary tumors and in bone metastases. Unexpectedly, blocking the pathway with ruxolitinib, or with the JAK2 inhibitor BSK805, enhanced the metastatic burden in both models. To investigate the effect of JAK inhibition on tumor cell dissemination from the primary site, we employed the 4T1.2 model, which spontaneously metastasizes from the mammary tumor to the bone and the lungs. As seen with the other models, there was a significant increase in tumor cell numbers in the bone and in the lungs in response to JAK inhibitor treatment. To understand the mechanism underlying the increase in metastatic load, we considered the host immune system as a potential bystander target of the JAK inhibitors. Indeed, in response to JAK inhibitor treatment we observed a major reduction in the NK cell population. The effect of JAK inhibitors on NK cells was systemic since they were also reduced in the bone marrow and in the peripheral blood of tumor-bearing mice. To mechanistically explain the impact of JAK inhibitors on NK cells, we used the NK cell line, NK-92. Upon treatment of NK-92 cells with JAK inhibitors, activation of multiple STATs decreased and cell proliferation was strongly inhibited. In cytotoxic assays, treatment with JAK inhibitors significantly decreased the killing ability of NK-92 cells against carcinoma cells. To test the in vivo relevance of NK cells in metastatic growth we used NSG mice, which are devoid of NK cell activity. Remarkably, in contrast to the results obtained with 4T1.2 mammary tumors grown in immunocompetent hosts, no increase in bone or lung metastases was observed in tumor-bearing NSG hosts treated with ruxolitinib, providing strong evidence that JAK inhibition increases metastasis by interfering with NK cell mediated anti-tumor innate immunity. These results suggest that the immunomodulatory effect of JAK inhibitors in breast cancer patients undergoing clinical trials should be monitored. Moreover, our work highlights the importance of evaluating the effect of targeted therapy on cell populations in the tumor environment in order to predict and overcame bystander effects that might impact on therapy response. Citation Format: Nancy E. Hynes, Alessia Bottos, Jason Gill, Thomas Radimerski, Alexander Tzankov, Aleksandra Wodnar-Filipowicz. The immunomodulatory effect of JAK inhibitors enhances metastasis by impairing antitumor immunity in preclinical models of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-013. doi:10.1158/1538-7445.AM2015-LB-013