Background

Kaposi’s sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS) (Chang et al., 1994; Ganem, 2010; Mesri et al., 2010; Dittmer and Damania, 2016). KS is a major cancer associated with AIDS (AIDS-KS) and is the most prevalent type of cancer affecting men and children in Africa (Ganem, 2010; Mesri et al., 2010; Cavallin et al., 2014). Although the incidence of AIDS-KS in the western world has markedly declined since the wide-spread implementation of HAART (Highly Active Antiretroviral Therapy), a significant percentage of AIDS-KS patients never achieve total remission (Krown, 2003; Nguyen et al., 2008; Cavallin et al., 2014). Moreover, KSHV prevalence and KS incidence appear to be increasing, even in HAART treated HIV patients with controlled viremias (Maurer et al., 2007; Labo et al., 2015). Understanding the interplay of viral and host factors in KS oncogenesis is critical for the rational development of new therapies (Dittmer and Krown, 2007; Sullivan et al., 2009). Many host signaling cascades co-opted by KSHV including PI3K/AKT/mTORC, NFkB and Notch are critical for cell-specific mechanisms of transformation and their identification is paving the way to therapeutic target discovery (Sodhi et al., 2006; Emuss et al., 2009; Liu et al., 2010; Mesri et al., 2014; Dittmer and Damania, 2016). Analysis of the molecular KS signature common to human KS tumors and our mouse KS-like tumors, showed consistent expression of KS markers VEGFR 1, 2, 3, Podoplanin with upregulation of angiogenesis ligands and receptors in vivo, pointing to the upregulation of various receptor tyrosine kinase signaling axes (Mutlu et al., 2007; Mesri et al., 2010). Thus, we set out to rank the host tyrosine kinase signaling cascades activated by KSHV using a mouse model of KSHV-dependent tumorigenesis that helped us to identify PDGFRA as an oncogenic driver and therapeutic target in AIDS-KS (Cavallin et al., 2018).

Here we describe an assay protocol using a Mouse Phospho-RTK Array Kit in mouse KSHV-induced KS-like tumors, which can be used as a reliable method to rank the tyrosine kinase receptors most activated in tumors in an unbiased manner. The Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems) is a membrane-based sandwich immunoassay. Captured antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample. Tyrosine phosphorylation of the captured proteins is detected with an HRP-conjugated pan phospho-tyrosine antibody and then visualized using chemiluminescent detection reagents. The signal produced is proportional to the amount phosphorylation in the bound analyte.

Surprisingly, analysis of the pattern of tumor RTK activation (Figures 1 and 2) showed a single prominently activated RTK spot that accounted for a significant portion of the total tyrosine kinase activation of the tumors. This signal corresponded to the PDGF receptor alpha-chain (PDGFRA) (Cavallin et al., 2018). Validation of the array by Western Blot analysis (Figure 3A) shows that PDGFRA is robustly expressed and much more phosphorylated in the tumors than in mouse normal skin. On the other hand, c-kit (Figure 3B), a related RTK, is similarly expressed in skin and tumors and displays low levels of activation in the tumors; that are similar to those in the skin control and correlates with the low activation of c-kit in our phospho-tyrosine kinase proteomic array (Cavallin et al., 2018).