VEGFC and lymphangiogenesis in clear cell renal cell carcinomas: Beyond the Abstract

Clear cell renal cell carcinoma (RCC) represents 85% of kidney cancers and 3% of adult cancers. If diagnosed early, the main treatment is surgery. However, many patients are diagnosed in metastatic stage. Metastatic RCC (mRCC) patients have a poor prognosis because of intrinsic resistance to radio- and chemo-therapy. The main feature of RCC is hyper-vascularization explained by overexpression of vascular endothelial growth factor (VEGF), which is linked to mutation/inactivation of the von Hippel-Lindau (vhl) gene - an E3 ubiquitin ligase of the Hypoxia Inducible-Factor 1α (HIF-1α). The reference treatments for mRCC are anti-angiogenesis therapies.

Sunitinib, a multi kinase inhibitor (TKI) targeting VEGF, PDGF, CSF1 receptors, c-KIT, FLT3 and RET, was approved in the first-line setting, with a median PFS of 11 months 1. Other targeted therapies have been developed, including TKIs like axitinib 2 or pazopanib 3-4 and the  mTOR inhibitors everolimus 5 and temsirolimus 6. More recently, cabozantinib which inhibits VEGFR, c-MET and AXL and the anti-PD1 immune checkpoint inhibitor nivolumab were proposed as other alternatives7-8.

However, sunitinib treatment benefits are transient in most cases and the majority of patients relapse after one year 9

Perhaps most provocatively, recent preclinical evidence has shown that anti-angiogenic treatment may elicit metastatic cell phenotypes which, in turn, may also compromise tumor-reducing benefits10-11. However, the results have been contradicted by clinical studies12.

Several studies have shown a close relationship between lymphangiogenesis, invasion and metastasis. During tumor development, the lymphatic system is considered one of the primary routes of tumor cell dissemination that leads to distant metastatic growth. VEGFC is currently the best characterized lymphangiogenic factor that acts via VEGFR3 13. In normal adult tissues VEGFR3 expression is largely restricted to lymphatic endothelial cells (LEC) and its activation is responsible for LEC proliferation, migration and survival. Several reports indicate that VEGFC expression in cancer cells correlates with lymphangiogenesis, accelerated tumor progression and/or a poor clinical outcome14.  

In our study15 we described the molecular mechanism linking sunitinib treatment to lymphatic vessels development through the stimulation of vegfc gene transcription and stabilization of VEGFC mRNA. Indeed, sunitinib activates the p38/MAP Kinase signalling pathway leading to increased i) transcription of the vegfc gene and ii) VEGFC mRNA half-life. The stability of VEGFC mRNA depends on a subtle balance of the mRNA binding proteins HuR and tristetraproline (TTP). Both proteins bind a specific AU rich (ARE) sequence in the 3' non-coding region of the VEGFC mRNA. These two binding proteins are considered as an oncogene (HuR) or as a tumor suppressor (TTP). HuR binding stabilizes target mRNA whereas TTP binding leads to their degradation (TTP)16. In addition, sunitinib stimulates the development of lymphatic vessels depending on VEGFC produced by tumor cells in experimental models of mRCC in mice. A lymphatic network was also observed in tumors from patients treated with sunitinib (presence of lymphatics in 70% of sunitinib-treated patients, vs 30% in untreated patients, Figure 1) in a neo-adjuvant setting (treatment given before surgery). An analysis of the fate of patients treated at the Cancer Centre of Nice (Centre Antoine Lacassagne) showed a strong correlation between the use of sunitinib and the presence of lymph node metastases and the development of new metastatic sites. 

Whereas sunitinib inhibits VEGFR2 and VEGFR3, the action of VEGFC may happen during the intercure (four weeks of treatment interrupted two weeks to limit toxic effects). Alternatively, VEGFC may stimulate neuropilin-2 (NRP2), the co-receptor of VEGFR3, which is overexpressed on RCC cells17.

Moreover, we believe that the other newly developed TKIs will induce the same side effects, with development of lymphatic vessels and metastatic dissemination. Indeed, we have also shown that pazopanib and axitinib, like sunitinib, stimulated VEGFC expression by the tumor cells via the activation of the p38/MAP Kinase pathway. The effect of cabozantinib on the expression of VEGFC by RCC cells remains to be determined.

Our findings suggest that anti-angiogenic treatment benefits may be compromised by the treatment-dependent development of lymphatic vessels. The subsequent pro-metastatic behaviour may compromise treatment efficacy.

We found an increase of lymphatic vessel in tumors from patients treated with sunitinib (70%). However, lymphatic vessels were also observed in 30% of untreated patients that were either metastatic or not (Figure 1). The determination of the aggressiveness of their cancers according to the presence of lymphatic vessels may be used for a better stratification of at-risk patients. A specific follow-up would improve the therapeutic reactivity for a better outcome. 

By in silico analysis, we have shown that high levels of VEGFC mRNA are associated with poor prognosis in RCC. Do patients presenting intra-tumor lymphatic vessels, strongly expressed VEGFC?

We conducted a clinical study to determine if VEGFC plasmatic level at the diagnosis, in mRCC patients, could represent a prognostic marker of progression free or overall survival. Unfortunately, we did not observe a specific correlation between VEGFC plasmatic amounts and survival. Trapping of VEGFC produced by cancer cells in the tumor without any diffusion in the bloodstream may explain this discrepant result. Moreover, VEGFA plasmatic amount is not correlated either with survival.

Despite these conflicting results, we are convinced that VEGFC remains prognostic marker of shorter survival but its presence should be evaluated in the tumor. As a complementary analysis, the presence of lymphatic vessels should also be evaluated in the primary tumor and correlated with both overall and progression-free survival. We hypothesize that the time to progression of non-metastatic patients presenting lymphatic vessels in their tumors will be shorter.

The involvement of lymphatic vessels in the aggressiveness of RCC needs to be addressed more in depth. Moreover, combining VEGFC inhibitors with sunitinib, or other TKIs, could limit the development of lymphatic vessels and keep tumor growth and dissemination in check.

Written By: Maeva Dufies, MD, University of Nice Sophia Antipolis, Institute for research on cancer and aging of Nice, CNRS UMR 7284; INSERM U1081, Centre Antoine Lacassagne, France;   Damien Ambrosetti, MD, PhD, Centre Hospitalier Universitaire (CHU) de Nice, Hôpital Pasteur, Central laboratory of Pathology;   Renaud Grépin, MD, Centre Scientifique de Monaco, Biomedical Department, 8 Quai Antoine Ier, MC-98000 Monaco, Principality of Monaco;   Gilles Pagès, PhD, University of Nice Sophia Antipolis, Institute for research on cancer and aging of Nice, CNRS UMR 7284; INSERM U1081, Centre Antoine Lacassagne, France

This work was supported by the French association for cancer research (ARC), the French National Institute for Cancer Research (INCA), the Fondation de France, the Fondation d'entreprise Groupe Pasteur Mutualité, "Cordon de Vie" Monaco”, and the Iris Pharma company.

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1. Motzer, R. J. et al. Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 27, 3584-3590, doi:JCO.2008.20.1293 [pii]10.1200/JCO.2008.20.1293 (2009).
2. Motzer, R. J. et al. Axitinib versus sorafenib as second-line treatment for advanced renal cell carcinoma: overall survival analysis and updated results from a randomised phase 3 trial. Lancet Oncol 14, 552-562, doi:S1470-2045(13)70093-7 [pii]10.1016/S1470-2045(13)70093-7 (2013).
3. Escudier, B. et al. Randomized, controlled, double-blind, cross-over trial assessing treatment preference for pazopanib versus sunitinib in patients with metastatic renal cell carcinoma: PISCES Study. J Clin Oncol 32, 1412-1418, doi:JCO.2013.50.8267 [pii]10.1200/JCO.2013.50.8267 (2014).
4.. Motzer, R. J. et al. Pazopanib versus sunitinib in metastatic renal-cell carcinoma. N Engl J Med 369, 722-731, doi:10.1056/NEJMoa1303989 (2013).
5. Motzer, R. J. et al. Phase 3 trial of everolimus for metastatic renal cell carcinoma : final results and analysis of prognostic factors. Cancer 116, 4256-4265, doi:10.1002/cncr.25219 (2010).
6. Motzer, R. J. et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet 372, 449-456, doi:S0140-6736(08)61039-9 [pii] 10.1016/S0140-6736(08)61039-9 (2008).
7. Motzer, R. J. et al. Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med 373, 1803-1813, doi:10.1056/NEJMoa1510665 (2015).
8. Choueiri, T. K. et al. Cabozantinib versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med 373, 1814-1823, doi:10.1056/NEJMoa1510016 (2015).
9. Escudier, B. Sunitinib for the management of advanced renal cell carcinoma. Expert review of anticancer therapy 10, 305-317, doi:10.1586/era.10.26 (2010).
10. Ebos, J. M. et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15, 232-239, doi:10.1016/j.ccr.2009.01.021 (2009).
11. Paez-Ribes, M. et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15, 220-231, doi:10.1016/j.ccr.2009.01.027 (2009).
12. Blagoev, K. B. et al. Sunitinib does not accelerate tumor growth in patients with metastatic renal cell carcinoma. Cell Rep 3, 277-281, doi:S2211-1247(13)00022-3 [pii] 10.1016/j.celrep.2013.01.015 (2013).
13. Alitalo, A. & Detmar, M. Interaction of tumor cells and lymphatic vessels in cancer progression. Oncogene 31, 4499-4508, doi:10.1038/onc.2011.602 (2012).
14. Su, J. L. et al. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer 96, 541-545, doi:10.1038/sj.bjc.6603487 (2007).
15. Dufies, M. et al. Sunitinib stimulates expression of VEGFC by tumor cells and promotes lymphangiogenesis in clear cell renal cell carcinomas. Cancer Res, doi:10.1158/0008-5472.CAN-16-3088 (2017).
16. Griseri, P. & Pages, G. Regulation of the mRNA half-life in breast cancer. World journal of clinical oncology 5, 323-334, doi:10.5306/wjco.v5.i3.323 (2014).
17. Cao, Y. et al. Neuropilin-2 promotes extravasation and metastasis by interacting with endothelial alpha5 integrin. Cancer Res 73, 4579-4590, doi:10.1158/0008-5472.CAN-13-0529 (2013).