The primary or in transit site

The primary or in-transit site of metastasis for many cancers, including melanoma, are nearby sentinel lymph nodes (LNs) [6]. As such, sentinel LN biopsy is a common procedure to diagnose and stage melanoma progression [7,8]. Sentinel LNs are often the primary site of metastasis due to tumor cell-stromal cell interactions that lead to increased expression of growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-B, that promote lymphangiogensis to the primary tumor [9]. As a tissue with a high rate of immune cell trafficking, LNs express several cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecules (ICAMs), that slow down and arrest immune cells. However, the exact mechanism of melanoma metastasis to lymph nodes remains unknown. Focal adhesion kinase (FAK) is a protein tyrosine kinase that is activated via integrins and growth factor receptors [10]. Activation of FAK plays a critical role in cellular migration and proliferation as well as tumor metastasis and angiogenesis [11,12]. A FAK kinase-dead knock-in mouse was found to be embryonic lethal at E9.5 due to loss of VCAM-1, which is critical for chorion-allantois fusion [13]. Recently, we found that pharmacological FAK inhibition also reduced proinflammatory VCAM-1 expression in human endothelial Cidofovir (ECs) [14]. Since LNs constantly express VCAM-1 [15] and most melanomas that express α4 integrin (a ligand for VCAM-1) showed greater metastatic potential to regional LNs [16,17], we hypothesized that FAK inhibition could reduce VCAM-1 expression in LNs and thus block metastasis of α4 integrin-expressing melanoma to LNs. In this study, we used the α4 integrin-expressing murine B16F10 melanoma to test pharmacological FAK inhibition (PF-271) on VCAM-1 expression and melanoma metastasis to LNs. Both in vitro and in vivo FAK inhibition reduced VCAM-1 expression in lymphatic ECs. Furthermore, FAK inhibition reduced B16F10 adhesion to and migration through human dermal lymphatic ECs. Finally, using a mouse footpad metastasis model, we found that FAK inhibition effectively diminished B16F10 melanoma metastasis to LNs by reducing FAK activity and VCAM-1 expression in lymphatic vessels. Taken together, our data demonstrate that pharmacological FAK inhibitors may provide a potential treatment option for initial metastasis to sentinel LNs.
Methods
Results
Discussion The process by which tumor cells metastasize from a primary lesion to a distant tissue is a complex process that requires several successful steps: 1) intravasation, 2) adherence to the endothelium and subsequent extravasation, and 3) creating a new niche at the site of metastasis [20]. Although 90% of cancer deaths are due to metastasis [21], the molecular mechanisms of metastasis are still not fully defined. Here, we showed that inhibition of FAK activity both in vitro and in vivo decreased VCAM-1 expression in lymphatic ECs (Fig. 1, Fig. 2). In vitro, FAK inhibition reduced B16F10-RFP cell adhesion and transmigration on HDLECs upon TNF-α stimulation (Fig. 1). Furthermore, FAK activity is critical for constitutive VCAM-1 expression in the lymphatic vessels of LNs (Fig. 2). Additionally, treatment with PF-271 reduced B16F10-RFP cell metastasis to popliteal LNs (Fig. 3, Fig. 4). While most trials for FAK inhibitors in clinical studies are focused on tumor cell proliferation and augmenting tumor cell apoptosis [22,23], our current study has shed new insights into the correlation between FAK activity-mediated changes in potential metastatic sites and tumor metastasis. Most notably, FAK activity regulating VCAM-1 expression in LNs raises the possibility that small molecule FAK inhibitors may be a novel preventative option for metastatic cancers that express α4 integrin, but not limited to melanoma [24,25]. As adherence to the endothelium and subsequent extravasation into the surrounding tissue are key steps in metastasis, several studies have investigated the role cell adhesion molecules play in this process. Expression of cell adhesion molecules, such as ICAM-1 and E-selectin, in certain tissues seem to play an important role in their prevalence as sites of metastasis. ICAM-1 plays a role in promoting metastasis to the liver [26], and ICAM-1 knockdown reduced B16F10 metastasis to the liver in mice [27]. FAK activity was shown to regulate E-selectin expression, which was important for metastasis to the lungs [28]. It will be intriguing to test if FAK activity not only regulates expression of these different cell adhesion molecules in LNs, but also in tissues with a high rate of metastasis.