FLT 1 kinase is a mediator of radioresistance and survival in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the fi fth most common malignancy worldwide, responsible for approximately half a million new cases every year. The treatment of this disease is challenging and characterised by high rates of therapy failure and toxicity, stressing the need for new innovative treatment strategies. Material and methods. In this study we performed a shRNAmir-based screen on HNSCC cells with the aim to identify tyrosine kinases that are mediating radiotherapy resistance. Results. The receptor tyrosine kinase FLT1 (VEGFR1) was identifi ed as an important driver of cell survival and radioresistance. We show that FLT1 is phosphorylated in HNSCC cells, and document autocrine production of FLT1 ligands VEGFA and VEGFB, leading to receptor activation. Immunohistochemistry on HNSCC patient samples demonstrated FLT1 and VEGFA to be uniformly expressed. Interestingly, FLT1 was selectively overexpressed in tumour tissue as compared to non-cancerous epithelium. Remarkably, we found only membrane permeable FLT1 kinase inhibitors to be effective, which was in agreement with the intracellular localisation of FLT1. Discussion and conclusion. Taken together, we document expression of FLT1 in HNSCC and demonstrate this kinase to modulate radioresistance and cancer cell survival. Given the fact that FLT1 kinase is selectively upregulated in tumour tissue and that its kinase function seems expendable for normal life and development, this kinase holds great promise as a new potential therapeutic target. The radiotherapy treatment for advanced stage head and neck squamous cell carcinoma (HNSCC) is challenging and characterised by high rates of therapy failure and toxicity. Consequently, the interest for smart molecular tumour targeting strategies is high as they may improve cure rates without adding more toxicity. Therapeutic targeting of the tyrosine kinase EGFR is an example of such an approach, recently proven to be successful in clinical practice [1]. However, the therapeutic potential of other tyrosine kinases in HNSCC remains uncharacterised. In order to tackle this issue we performed an unbiased systematic micro-RNA-based genetic dropout screen to identify additional therapeutic targets in this large family of proteins. In this work, we identify and validate FLT1 (or VEGFR1) as a new target to modify HNSCC survival and radiosensitivity. FLT1 forms together with FLK (VEGFR2) and FLT4 (VEGFR3) the VEGF-receptor tyrosine kinase family. These receptors consist of seven extracellular immunoglobulin-like domains, a trans-membrane region and a tyrosine kinase domain [2]. FLT1 binds VEGFA, VEGFB and PlGF, whereas FLK binds VEGFA and VEGFC. The role of FLK (and VEGFA) as the primary driving force of angiogenesis is well established. The role of FLT1 in this context is more elusive. The current belief is that FLT1 primarily acts as a decoy receptor, reducing FLK activity by sequestering stimulatory VEGFA. Consistent with this model, FLT1 has been shown to bind VEGFA with much higher affi nity than FLK, but shows much less tyrosine kinase activity [2,3]. In accordance, loss of FLT1 expression causes embryonic lethality as a result of vascular overgrowth, while FLT1 kinase dead variants with normal VEGFA binding capacity Acta Oncologica, 2014; 53: 637–645 ISSN 0284-186X print/ISSN 1651-226X online © 2014 Informa Healthcare DOI: 10.3109/0284186X.2013. 835493 638 E. J. Van Limbergen et al. do show normal vascular development and good health [4,5]. Other evidence, however, indicates that the role of FLT1 in angiogenesis is more complex, as several reports have demonstrated that FLT1-mediated intracellular signalling regulates angiogenesis in several pathological conditions, one of which is cancer [3]. In this context, numerous authors have shown that FLT1 inhibition suppresses tumour growth and metastasis [3]. More recent reports also demonstrate FLT1 expression directly on tumour cells from breast, colon and skin origin, and show it to be an important oncogenic driver in these cells promoting survival, cell proliferation, invasiveness and/or motility, in an angiogenesis-independent manner [6 – 9]. In this paper we examine the role of FLT1 in HNSCC. Material and methods