There were five main findings of this study. First, the preoperative serum level of IFNAR2 mRNA was correlated with tumor size. Second, the tumor tissue levels of IFNAR2 mRNA and phosphorylated S6 ribosomal protein (Ser-235/236) were positively correlated with each other, and both were related to tumor metastatic potential. Third, while the patients with a high preoperative serum level of IFNAR2 mRNA showed a good response to IFN-α ± sorafenib (IFN-α ± Sor; CR-PR) and a longer overall survival, the patients with a low serum level and a high tumor tissue level of IFNAR2 mRNA level showed a poor response (SD-PD) and a shorter overall survival. Fourth, the patients with a high serum level of IFNAR2 mRNA throughout treatment showed a good response to IFN-α ± Sor. Fifth, a high level of phosphorylated Akt (Ser-473), but not a high level of phospho-S6 ribosomal protein (Ser-235/236), in the primary tumor was related to a poor response to IFN-α ± sorafenib. These findings suggest that the serum level of IFNAR2 mRNA might be useful to predict the efficacy of IFN-α ± sorafenib therapy, while the tumor tissue level of IFNAR2 mRNA could be associated with metastatic potential and tumor resistance.
Combination therapy with IFN-α and sorafenib
While the efficacy of combination therapy with IFN-α and sorafenib needs clarification, the adverse effects of this regimen are related to the doses of each agent and are not additive [
22‐
25]. We previously reported a good response to combination therapy with IFN-α and half the usual dose of sorafenib (400 mg/day rather than 800 mg/day) in patients with IFN-α-resistant RCC, along with tolerable adverse events [
18].
It has also been reported that combined treatment with IFN-α + sorafenib suppresses proliferation and vascular endothelial growth factor (VEGF) production by several RCC cell lines more strongly than either agent alone [
26,
27]. RCC is considered to be an immunogenic tumor [
3], since cytotoxic T lymphocytes recognize and selectively kill autologous RCC cells, while tumor-specific T cells can be detected in the blood of RCC patients [
3]. Sorafenib is a multikinase inhibitor targeting VEGF receptors 1–3, PDGFβ receptor, and Raf kinase, and it has both direct antitumor activity and antiangiogenic activity [
28]. In a randomized phase III trial comparing sorafenib with placebo as second-line therapy for RCC, the response rate to sorafenib was 10%, and the stable disease rate was 74%, with the median progression-free survival time being 5.5 months in the sorafenib group versus 2.8 months in the placebo group [
29]. In addition to its immunomodulatory effects, IFN-α also has direct antitumor activity as well as antiangiogenic activity, including inhibition of VEGF [
30]. Antitumor immunity is usually suppressed in tumor-bearing mice because of the influence of regulatory T cells and suppressive cytokines, such as TGF-β and IL-10 [
31]. Takeuchi et al. recently reported that the synergistic effect of sorafenib and IFN-α on RCC both in vitro and in tumor-bearing mice was related to a combination of antitumor, antiangiogenic, and immunologic responses [
27]. In their study, sorafenib had no effect on the levels of natural killer (NK) cells, T cells, and regulatory T cells in the spleens of tumor-bearing BALB/c mice, irrespective of the use of IFN-α, while IFN-α showed weaker direct antitumor activity than sorafenib but stimulated CTL, NK cells, and tumor-infiltrating lymphocytes (which sorafenib did not), so that a synergistic antiproliferative effect of these two agents was demonstrated in vitro [
27].
Although an additive effect of IFN-α to sorafenib therapy in patients with metastatic RCC was recently reported [
24], the clinical efficacy of this combination remains to be confirmed. Jonasch et al. reported that the outcome after addition of IFN-α to sorafenib was comparable to that of sorafenib monotherapy, when patients were randomized to treatment with either sorafenib (400 mg twice daily) or the combination of sorafenib (400 mg twice daily) plus IFN-α (0.5 MU twice daily) [
25]. However, their IFN-α dose of 1 million units daily was probably too low to assess its additive effect because the average dose is 3–9 million units daily. So far, the published studies on combination therapy with IFN-α and sorafenib have employed concomitant therapy with both agents. In contrast, our treatment strategy was to add sorafenib (400 mg/day) to IFN-α in RCC patients whose tumors were refractory to IFN-α alone, i.e., first-line IFN-α monotherapy and second-line combination therapy with IFN-α plus sorafenib [
18]. In the present study, 26 patients had metastatic disease (M1) at diagnosis. Among them, five patients showed a good response to IFN-α alone (IFN-α: CR-PR), 11 patients showed a good response to IFN-α + sorafenib (IFN-α + Sor: CR-PR, Table
1), and the remaining 10 patients had stable disease or showed a poor response to IFN-α + sorafenib (IFN-α + Sor: SD-PD, Table
1). Although half-dose sorafenib (400 mg/day) caused grade 1/2 toxicity, grade 3/4 toxicity was rare in the present study and such toxicity resolved when patients suspended sorafenib therapy. The response rate to the combination of IFN-α plus half-dose sorafenib was 52.4% (11/21 patients resistant to previous IFN-α monotherapy achieved CR or PR), indicating that sorafenib may be synergistic with IFN-α, leading to an increase of antitumor activity. Furthermore, the patients with good response to this combination therapy had favorable prognosis (Fig.
6). Thus, this combination seems to be tolerable and could be a useful treatment option for advanced RCC resistant to IFN-α monotherapy [
18].
Role of the IFNAR2 and mTOR pathways in progression of RCC
The preoperative serum level of IFNAR2 mRNA was not correlated with the effect of IFN-α monotherapy, but a lower tumor tissue level of IFNAR2 mRNA was related to a better response to IFN-α. The present finding that a higher tumor tissue level of IFNAR2 mRNA was associated with a poor response to IFN-α is consistent with our previous results [
14]. In contrast, regarding the relationship between IFNAR2 mRNA levels and the effect of combination therapy with IFN-α plus sorafenib, the preoperative serum level of IFNAR2 mRNA was higher in the IFN-α + Sor: CR-PR group and the IFN-α: CR-PR group than in the IFN-α + Sor: SD-PD group (Fig.
6e), while the tumor tissue level of IFNAR2 mRNA was lower in the IFN-α + Sor: CR-PR group and the IFN-α: CR-PR group than in the IFN-α + Sor: SD-PD group (Fig.
6f). These findings suggested that a different molecular mechanism might be involved in IFN-α ± Sor: CR-PR group and IFN-α + Sor: SD-PD group.
IFNs are pleiotropic cytokines that regulate antiviral, antitumor, apoptotic, antiangiogenic, and cellular immune responses via activation of multiple downstream signaling cascades, including the Janus tyrosine kinase (Jak)-signal transducer and activator of transcription (STAT) pathway, the p38 mitogen-activated protein kinase (MAPK) pathway, and the mTOR pathway [
32,
33]. The Jak-STAT and p38-MAPK signaling pathways have been shown to be responsible for transcription of the genes encoding proteins related to the antiviral and/or antiproliferative effects of IFNs. On the other hand, it has been reported that activation of the mTOR pathway by IFNs has an important regulatory role in mRNA translation and induction of the interferon response [
34,
35]. IFN-α-induced tumor cell apoptosis is also mediated via the mTOR pathway in a nucleus-independent manner [
36,
37]. Moreover, the Jak-STAT and mTOR pathways act separately from each other after activation by IFN-α [
37]. Thus, it is likely that mTOR signaling selectively mediates apoptosis and survival.
The rapamycin-sensitive mTOR-raptor (regulatory-associated protein of mTOR) complex controls cell growth by regulating protein synthesis, so mTOR-raptor signaling is a potential antitumor target, and mTOR inhibitors are currently under investigation for the treatment of various human cancers. On the other hand, mTOR also interacts with rictor (rapamycin-insensitive companion of mTOR), and recent findings have suggested that the rapamycin-insensitive effect of mTOR on cell survival is overactive in many cancers. Thus, mTOR has dual rapamycin-sensitive (mTOR-raptor complex: mTORC1) and insensitive (mTOR-rictor complex: mTORC2) functions, indicating that treatment with rapamycin will not completely inhibit mTOR activity [
38,
39]. Phosphatidylinositol 3`kinase (PI3 K), serine/threonine kinase Akt, and the mTOR pathway are all overactive in human cancers. mTORC1 lies downstream of PI3 K and is part of a pathway that is frequently activated in human cancers, so mTORC1 represents a pivotal target for anticancer therapy. The best-characterized pathways regulated by mTORC1 are phosphorylation and activation of ribosomal S6 kinase-1 (S6K1) and phosphorylation and inactivation of 4E-BP1, the suppressor of mRNA cap-binding protein eIF4E, leading to effects on cell growth and metabolism by acting as a restriction point in cells subjected to stresses [
40,
41] such as hypoxia [
42‐
44].
Phosphorylation at two sites is required for full activation of Akt, since it is phosphorylated by PI3 K-dependent kinase-1 (PDK1) at a threonine residue in the catalytic domain (Thr 308) and by PI3 K-dependent kinase-2 (PDK2) at a serine residue (Ser 473) in the carboxy-terminal hydrophobic motif [
45]. It has been reported that mTORC2 regulates the actin cytoskeleton and also possesses PDK2 activity that phosphorylates Ser-473 in the carboxy-terminal of Akt, making it essential for Akt activity [
46]. Importantly, activation of Akt may lead to cell survival when mTORC1 is inhibited or could potentially increase VEGF production because PI3 K/Akt signaling induces tumor angiogenesis by regulating VEGF via both HIF-1α-dependent and -independent mechanisms [
47]. It has been reported that hypoxia-inducible factor (HIF) 1α expression is dependent on both raptor and rictor, whereas HIF2α expression only depends on rictor and HIF2α is more important in RCC [
48]. These findings suggest that phosphorylation of Ser 473 in AKT is a key molecular step in the progression of RCC and could be a target for treating these tumors [
25]. In agreement with this, our current study showed that the tumor tissue levels of IFNAR2 mRNA and phosphorylated S6 ribosomal protein (Ser-235/236) were positively correlated and that both were related to tumor metastatic potential, while a high phosphorylated Akt (Ser-473) level in the primary tumor was associated with a poor response to IFN-α ± sorafenib therapy (stable or progressive disease).
Current efforts to achieve the clinical development of mTOR inhibitors are based on the role of mTOR signaling in promoting the proliferation and survival of tumor cells. It has been reported that treatment with mTOR inhibitors can improve the outcome of patients with metastatic RCC [
8‐
10]. On the other hand, the mTOR pathway is also important for IFN-dependent translational responses, and IFN-α is widely used to treat advanced RCC. Although we could not exclude a possible detrimental effect of IFN-α treatment in the patients with metastatic RCC and higher IFNAR2 mRNA levels in their tumors, our findings suggested that there may be different molecular mechanisms of cancer progression in the patients with a good or poor response to IFN-α ± sorafenib. It is possible that IFNAR2 signaling has different biological effects from normal when upregulated in RCC.
Jonasch et al. recently reported that an increase of phosphorylated Akt (Ser-473) was associated with worse survival by microarray analysis of paraffin-embedded specimens [
25]. In the present study, the tumors with higher phosphorylated Akt (Ser-473) levels, but not higher phospho-S6 ribosomal protein (Ser-235/236) levels, were resistant to IFN-α ± sorafenib therapy. In addition, the tumor tissue levels of IFNAR2 mRNA and phosphorylated S6 ribosomal protein (Ser-235/236) were positively correlated, and both were related to metastatic potential. Tumor levels of IFNAR2 mRNA also had a weak positive correlation with those of phosphorylated Akt (Ser-473). Moreover, the patients whose tumors had higher levels of phosphorylated Akt (Ser-473), phosphorylated S6 ribosomal protein (Ser-235/236), and IFNAR2 mRNA showed shorter overall survival. Taken together, it is possible that phosphorylation of Ser 473 on Akt is a key molecular step in the progression of RCC and a potential therapeutic target, so that the tumor level of phosphorylated Akt (Ser-473) may be useful for predicting the response to treatment. At present, it remains to be elucidated why upregulation of IFNAR2 expression is linked to the progression of RCC and to a poor response to treatment, and it is unclear how IFNAR2 interacts with mTORC1 and mTORC2, but our findings suggested that the IFNAR2-mTORC1 pathway via phosphorylated S6 ribosomal protein (Ser-235/236) may act locally within tumors to promote proliferation and metastasis by modifying mRNA translation, while the IFNAR2-mTORC2 pathway via phosphorylated Akt (Ser-473) may be associated with tumor resistance. So, these interactions should be elucidated in the future. As Lekmine et al. have indicated, therefore, caution should be exercised when designing clinical trials that combine an mTOR inhibitor and IFN-α due to possible antagonism of antitumor activity [
34]. In fact, Huges et al. reported that patients treated with temsirolimus alone had better overall survival than those given IFN-α alone, while patients treated with temsirolimus plus IFN-α did not [
49]. In the future, the downstream targets of IFNAR2 should be identified, and the expression or activity of one or two such targets should be studied in cell lines or tissue samples. A better understanding of the IFNAR2 pathway may help to elucidate its role in cancer.
Although none of our patients were treated by sorafenib alone, it would be interesting to assess the expression of not only IFNAR2, phosphorylated Akt (Ser-473), and phosphorylated S6 ribosomal protein (Ser-235/236), but also VEGF receptor and Raf, in tumor cells and the effects of sorafenib, sunitinib, or mTOR inhibitors. Such information could lead to elucidation of the role of the IFNAR2-mTOR pathway in the progression of RCC and the selection of patients who will benefit from treatment with IFN-α, sorafenib, sunitinib, or mTOR inhibitors.
Role of serum IFNAR2 in progression of RCC
The serum CRP level is associated with the stage and outcome of RCC [
50,
51]. Elevation of CRP is primarily determined by an increase of circulating IL-6 [
52], and the IL-6 level is correlated with the serum CRP level as well as with tumor histological grade and tumor metastasis [
53]. We previously reported that increased serum levels of CRP and IL-6 were associated with local tumor invasion and metastasis [
54]. In the present study, a higher preoperative serum CRP level was associated with local invasion and metastasis of RCC, but not with the response to treatment (data not shown). These findings suggest that the serum CRP level is associated with tumor aggressiveness, so that elevation of CRP might reflect the poorer general condition of the patient rather than the response to therapy. On the other hand, IFN-α has immunomodulatory effects and direct antitumor activity as well as antiangiogenic activity, including inhibition of VEGF [
30]. Therefore, it is unclear exactly what serum IFNAR2 reflects, but it is likely to be associated with the overall immune status. Accordingly, we expected to find a relation between the preoperative serum levels of IFNAR2 mRNA and CRP, but no relation was observed.
Although we could not examine how IFN-α binds to IFNAR2 on peripheral blood cells and then exhibits antitumor and antiangiogenic activity, our findings showed that the preoperative serum level of IFNAR2 mRNA was positively correlated with tumor size and was higher in patients with metastatic RCC who showed a good response to IFN-α ± sorafenib therapy than in those with a poor response. Because obtaining blood samples from patients is easier than harvesting tissue samples, chronological analysis of serum IFNAR2 mRNA levels is preferable for evaluation of the role of IFNAR2. While the effect of IFN-α therapy on the serum level of IFNAR2 mRNA is still unclear, our chronological evaluation of IFNAR2 mRNA throughout treatment showed that its serum level remained higher in the IFN-α ± Sor: CR-PR group than in the IFN-α + Sor: SD-PD group. Taken together, these observations suggest that an increased serum level of IFNAR2 mRNA may represent a systemic immunologic and antitumor response to the tumor burden in RCC patients, as well as showing antimicrobial activity if infection occurs.
Regarding the effect of genetic polymorphism on the response of metastatic RCC to IFN-α, it has been reported that STAT3 polymorphism is a useful diagnostic marker for predicting the response to IFN-α therapy in these patients [
55]. An efficient marker of the response to IFN-α is needed to establish individualized optimal treatment strategies, especially when newer therapies are used as first-line treatment for metastatic RCC. Our study showed that patients with higher serum levels of IFNAR2 mRNA may be more likely to respond to IFN-α ± sorafenib therapy and to show a longer overall survival, while patients with higher tumor tissue levels of IFNAR2 mRNA may show poor response and unfavorable overall survival. Although we need a surgical specimen to examine tumor tissue levels of IFNAR2 mRNA and protein, the serum level of IFNAR2 mRNA can be conveniently measured, so it may be more useful for predicting the response to IFN-α ± sorafenib therapy and as a prognostic indicator.