Sunitinib prevents cachexia and prolongs survival of mice bearing renal cancer by restraining STAT3 and MuRF-1 activation in muscle.

Tyrosine kinase inhibitors, affecting angiogenesis, have shown therapeutic efficacy in renal cell carcinoma (RCC). The increased overall survival is not fully explained by their anti-tumor activity, since these drugs frequently induce disease stabilization rather than regression. RCC patients frequently develop cachectic syndrome. We used the RXF393 human renal carcinoma xenograft that recapitulates the characteristics of the disease, including the growth in the mouse kidney (orthotopic implantation), and the induction of cachexia with subsequent premature death. Sunitinib prevents body weight loss and muscle wasting and significantly improves the survival of RXF393-bearing nude mice. The anti-cachectic effect was not associated to direct anti-tumor activity of the drug. Most relevant is the ability of sunitinib to reverse the cachectic phenotype and rescue the animals from the loss of fat tissue. Body weight loss is prevented also in mice bearing the C26 colon carcinoma, classically reported to induce cachexia in immunocompetent mice. Among the mechanisms, we herein show that sunitinib is able to restrain the overactivation of STAT3 and MuRF-1 pathways, involved in enhanced muscle protein catabolism during cancer cachexia. We suggest that off-target effects of angiogenesis inhibitors targeting STAT3 are worth considering as a therapeutic option for patients who develop cachexia, independently of their anti-tumor activity.

Chemotherapy counteracts metastatic dissemination induced by antiangiogenic treatment in mice.

The development of resistance and progressive disease after treatment with angiogenesis inhibitors is becoming a controversial issue. We investigated the experimental conditions that cause multireceptor tyrosine kinase inhibitors (RTKI) to augment metastasis and whether opportune combinations with chemotherapy could counteract this effect. The renal Renca-luc tumor was transplanted orthotopically in the kidney of Balb/c mice, which then were or were not nephrectomized. The Lewis Lung carcinoma (LLC) was transplanted in the tibial muscle of C57/Bl6 mice. Treatment with the RTKI sunitinib started at different stages of tumor progression, mimicking neoadjuvant or adjuvant settings. Combination studies with paclitaxel, doxorubicin, cisplatin, gemcitabine, and topotecan were done on the LLC model, using opportune regimens. In a neoadjuvant setting, sunitinib inhibited Renca-luc tumor growth, prolonging survival despite an increase in lung metastasis; treatment after primary tumor surgery (adjuvant setting) or on established metastasis prolonged survival and decreased metastasis. Sunitinib increased lung metastasis from mice bearing early-stage LLC, but did not affect established metastases (no acceleration) from advanced tumors. Combinations with doxorubicin, topotecan, gemcitabine, but not cisplatin and paclitaxel, counteracted the increase in metastasis from LLC, partly reflecting their antitumor activity. Histology analysis after sunitinib confirmed tumor vascular changes and increased hypoxia. Topotecan at suboptimal daily doses reduced sunitinib-related metastasis, reducing tumor hypoxia. Tyrosine kinase inhibitors, as sunitinib, can have adverse malignant effects mainly in the neoadjuvant setting. The addition of chemotherapy might influence metastasis, depending on each drug mechanism of action and its regimen of administration.

E-3810 is a potent dual inhibitor of VEGFR and FGFR that exerts antitumor activity in multiple preclinical models.

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF-induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation.

The effects of vandetanib on paclitaxel tumor distribution and antitumor activity in a xenograft model of human ovarian carcinoma.

This study was designed to determine the effects of vandetanib, a small-molecule receptor tyrosine kinase inhibitor of vascular endothelial growth factor and epidermal growth factor receptor, on paclitaxel (PTX) tumor distribution and antitumor activity in xenograft models of human ovarian carcinoma. Nude mice bearing A2780-1A9 xenografts received daily (5, 10, or 15 days) doses of vandetanib (50 mg/kg per os), combined with PTX (20 mg/kg intravenously). Morphologic and functional modifications associated with the tumor vasculature (CD31 and alpha-smooth muscle actin staining and Hoechst 33342 perfusion) and PTX concentrations in plasma and tumor tissues were analyzed. Activity was evaluated as inhibition of tumor growth subcutaneously and spreading into the peritoneal cavity. Vandetanib treatment produced no significant change in tumor vessel density, although a reduced number of large vessels, an increased percentage of mature vessels, and diminished tumor perfusion were evident. Pretreatment with vandetanib led to decreased tumor PTX levels within 1 hour of PTX injection, although 24 hours later, tumor PTX levels were comparable with controls. In efficacy studies, the combination of vandetanib plus PTX improved antitumor activity compared with vandetanib or PTX alone, with greater effects being obtained when PTX was administered before vandetanib. The combination of PTX plus vandetanib reduced tumor burden in the peritoneal cavity of mice and significantly increased their survival. Analysis of vascular changes and PTX tumor uptake in vandetanib-treated tumors may help to guide the scheduling of vandetanib plus PTX combinations and may have implications for the design of clinical trials with these drugs.

Impact of VEGF-dependent tumour micro-environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice.

Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.