Renal Cell Carcinoma RCC is a lethal cancer

Renal Cell Carcinoma (RCC) is a lethal cancer with bad prognosis due to development of chemoresistance and recurrence of an aggressive tumor with increased tumor-angiogenesis and metastasis [7]. We have found Axl and Gas6 to be differentially presented in RCC subtypes, and Axl to correlate to tumor advancement, and to add prognostic information to patient survival [8]. Inhibition of tumor-angiogenesis by targeting of VEGFRs and PDGFRs on endothelial NVP-AUY922 (EC) by the small molecule inhibitor Sunitinib is first-line treatment of advanced clear cell RCC (CCRCC). However, life improvement of treated patients is poor, and there is need for advancement of treatment options [7], [9]. As mentioned, Axl regulates multiple tumorigenic processes contributing to advanced disease. For instance, in tumor-angiogenesis the Gas6-Axl pathway governs EC migration and recruitment and sprouting of new vessels [10]. Axl is also involved in EMT, a cellular event rendering epithelial cells more mesenchymal-like and motile, and by this means Axl plays an important role in the metastatic process [11], [12], [13]. Administration of soluble Axl in circulation prevents metastasis of cancer cells, and knockdown of Axl in CCRCC tumor cells results in lower metastatic events [11], [12], [14]. Moreover, Axl is suggested to mediate achieved resistance in several cancers, for instance by “taking-over” tumor promoting signaling from targeted RTKs, or by diversification of targeted signaling pathways [15], [16], [17]. Axl is also involved in maintaining stemness and support of non-adhesive growth of cancer cells [11], [18], mechanisms strongly related to non-solid tumor cell survival, spread and repopulation of secondary organs [19]. Today, several Axl-specific inhibitors are under development. R428 is one with high Axl-selectivity and treatment with R428 inhibits tumor-angiogenesis and blocks tumor spread in vivo[20].
Material & methods
Results
Discussion Based on the results presented here we hypothesize that Sunitinib treatment, which is used to target EC cells and tumor-angiogenesis, provides a tumorigenic TME where Gas6 that is present in the tumor mass [28] stimulates Axl signaling, in both CCRCC tumor and EC cells, in an intensified, diversified and constitutive manner. In other words, by seeking health-improvement with Sunitinib treatment, a protumorigenic side effect originating from Sunitinib+Gas6 co-stimulation might be the end result of the treatment. We show that Gas6-mediated Axl and downstream AKT pathway activation is triggered and intensified by the Gas6+Sunitinib combination with phosphorylation of both pSer473 and pThr308 AKT sites and of the downstream AKT target PRAS40-Thr246. PRAS40-Thr246 is elevated in many cancers and predicts hyperactivation of the PTEN/PI3K/AKT pathway [25]. Increased AKT signaling in EC cells stimulates tumor-angiogenesis with formation of unstable leaky new blood vessels contributing to insufficient blood supply, hypoxia, increased metastasis and shortage of drug delivery to tumor core [39]. Remarkably, in a Sunitinib environment, Gas6-mediated Axl signaling is not only intensified but also diversified and results in onset activation of EGFR and HGFR and conserved MAPK pathways beyond those signaling pathways triggered by Gas6 alone. This is of great interest since these RTKs and MAPK pathways are all known to drive tumorigenesis and resistance development in many cancers, even in crosstalk with Axl [17], [27], [40], [41]. Altogether, this results in a tumor-promoting milieu affecting both tumor CCRCC and EC cells that might drive development of chemoresistance and recurrence of aggressive disease. Investigating optimal conditions for Gas6+Sunitinib cultures, we found that CCRCC cells had to be confluent for valid analysis of long-term Gas6-Axl-dependent mechanisms. Long-term cultures of confluent cells only displayed Gas6-dependent Axl and downstream pSer473AKT activation and increased viability and decreased apoptosis. Axl levels were reduced to half after long-term Gas6 stimulation of confluent cells. This is in contrast to subconfluent cells displaying an extensive Axl downregulation and degradation [28], [42]. Hence, we believe Axl expression to be rescued in confluent cells resembling a situation of dense tumor mass in which Axl is highly expressed and active [28]. Interestingly, hypoxia that often is occurring in the tumor core stabilizes Axl expression and avoids ligand-induced downregulation [43]. Comparing constitutive signaling in Gas6+Sunitinib-adapted cells with peak short-term signaling we found an intensified activation of Axl and downstream pSer473AKT in short-term conditions but no activation of pThr308AKT or divergent MAPK pathways indicating different Axl-dependent long-term and short-term signaling. This is in line with reported Axl-dependent conserved pSer473AKT activation and down-regulated ERK signaling in EGFR resistant cancer cells [40].