In this study, we characterized the levels of mRNAs encoding proteins involved in the IGF pathway in bladder cancers. We then assessed the correlation of these mRNA levels with tumor stage, activation of the INSR/IGF1R receptors (indicating activation of the IGF pathway) and the antiproliferative efficacy of an inhibitor of IGF1R tyrosine kinase.
We found that among the IGFBPs studied (IGFBP1–7), IGFBP7 is the only IGFBP down-regulated during tumor progression. This specificity is maybe linked to the fact that among these IGFBPs, IGFBP-7 is the only low affinity IGFs binding protein and is therefore more related to IGFBP8–10 that are part of the CCN family.
We found that IGF1R expression (mRNA and protein levels were) was higher in Ta tumors than in more invasive tumors and that
IGF1R mRNA expression in normal urothelium did not differ significantly from that in tumor samples. These findings, confirmed at the mRNA level in two other publicly available data sets, go against the conclusions of the study by Rochester et al., the only other study to date to have considered IGF1R expression as a function of bladder cancer stage [
9]. They found
IGF1R mRNA and IGF1R levels to be significantly higher in MIBC tumors than in normal bladder tissues, consistent with an upregulation of transcription. The discrepancies observed may result from the number of samples studied. For the transcriptomic analysis, Rochester et al. have studied 15 normal urothelium samples and 17 MIBC samples, while we have studied a total of 11 normal samples and 368 MIBC samples in our study. For the protein analysis, discrepancy could also be due to very different techniques: heterogeneity within the tumor is difficult to assess by immunohistochemistry. On the other hand, RPPA does not distinguish between protein expression in the tumor cells or in the stroma.
This study is the first to show that the activation of the IGF pathway is stronger in Ta tumors than in more invasive tumors (T1 and
T ≥ 2). The phosphorylation of INSR/IGF1R proteins, leads to the activation of two main signaling pathways: the PI3K-AKT/PKB pathway and the RAS-MAPK pathway. Interestingly, these two pathways are also activated by Fibroblast Growth Factor Receptor 3 (FGFR3) that is commonly mutated and constitutively activated in Ta tumors. The activation of these two receptors thus leads to the same proliferation signal, which is crucial in the oncogenesis of Ta G1/low-grade bladder cancers [
25]. Consistent with this hypothesis, CCLE cell viability assays with the IGF1R kinase inhibitor (AEW541) on 13 bladder cancer cell lines showed that cell lines derived from low-grade/stage tumors and also presenting a translocated form of FGFR3 (RT112, SW780, RT4) were among the most sensitive to the anti-proliferative effects of AEW541 (Fig.
4a).
Finally, we used a supervised approached towards known IGF pathway’s gene to identify molecular predictors of IGFR pathway activation (measured as IGF1R phosphorylation) in tumors and sensitivity to an inhibitor of IGF1R tyrosine kinase (AEW541) in bladder derived cell lines. Surprisingly, our results demonstrate that the expression levels of genes encoding IGF receptors and ligands were not correlated with the activation of INSR/IGF1R receptors or with the anti-proliferative efficacy of AEW541. However,
IGFBP5 mRNA levels in bladder cancer samples and cell lines were negatively correlated with the activation of INSR/IGF1R, and in cell lines with the anti-proliferative efficacy of an IGF1R tyrosine kinase inhibitor, suggesting that IGFBP5 over-expression in MIBC might be a useful marker of non-sensitivity to IGF1R inhibitor. Using the same hypothesis-driven supervised approach focused on IGF pathway member expression, Pavlicek and al., also identified IGFBP5 as a biomarker of colon cancer cells sensitivity to another IGF1R tyrosine kinase inhibitor, Figitumumab [
26]. Using an unsupervised approach that used the whole genome data, given the different kinds of cancers studied within the CCLE project, the sensitivity of cells lines to the IGFR kinase inhibitor AEW541 was also predicted by
IGFBP5 expression levels [
19]. Thus, it may be possible to extrapolate the results reported here for bladder cancers to other cancer types [
19]. At the opposite, others markers of IGFR inhibitor sensitivity identified previously [
19,
26] such as MYB did not predict sensitivity to AEW541 in bladder tumor derived cell lines and seem so to be cancer specific (data not shown). IGFBP5 is a carrier protein that may increase the half-life and the turnover in the bloodstream of IGFs [
27]. IGFBP5 can play various roles during tumor progression [
28], sometimes in absence of IGFs, supporting the existence of some IGF-independent activities [
29]. I
GFBP5 expression is altered in various cancers, including breast cancer, neuroblastoma, osteosarcoma, lung cancer, colon cancer, and its impact on prognosis has been shown to be cell type-dependent and tissue type-dependent [
27]. Liang et al. recently showed that
IGFBP5 overexpression was associated with a poor prognosis in patients with urothelial carcinomas of the upper urinary tract and urinary bladder [
30]. Using immunohistochemistry, these authors showed that IGFBP5 overexpression was significantly associated with advanced tumor stage, and that it was an independent predictor of poor disease-specific survival and metastasis-free survival. We report here that this over-expression in MIBC is also a marker of cell non-sensitivity to IGF1R inhibitor. Our results should increase interest in IGFBP5. Indeed, as part of the current focus of pharmacological research on the INSR/IGF1R pathway in the treatment of cancer, more than 100 clinical trials have already investigated INSR/IGF1R inhibition. Several studies testing anti-IGF1R monoclonal antibodies reported low toxicity, with major clinical responses in some cases [
31‐
33]. Nevertheless, the first phase III trial, which studied the addition of figitumumab to standard chemotherapy in the treatment of non-small-cell lung carcinoma was stopped after the inclusion of 682 patients because of toxicity, the signs of which included hyperglycemia in particular, together with a lack of antitumor efficacy [
32]. The results of other phase III studies are expected soon, but schisms are already evident: some teams have abandoned the development of treatments targeting INSR/IGF1R, whereas others are continuing to study these receptors, whilst searching, in parallel, for biomarkers of the effectiveness of these therapies. Our results suggest that IGFBP5 expression could be used in this way in the bladder cancer setting.