Background
Muscle-invasive bladder cancer (MIBC) refers to cancers happening in the muscle wall of the bladder. Symptoms such as pain with urination, blood in the urine, and low back pain are often observed in patients with bladder cancer. Bladder cancer is one of the most common malignancies worldwide [
1]. It is much more commonly diagnosed in men than in women, but female patients are usually with more advanced stages at the time of diagnosis, and exhibit less favorable survival [
2]. MIBC has the potential to spread to nearby lymph nodes and other organs. In severe cases, metastasis would affect distant organs such as lungs and liver [
3]. Increasing age is considered to be the main risk factor for bladder cancer, and impacts from smoking and exposure to some industrial chemicals are also reported to be significant [
4].
With the advances in high-throughput technologies, several prognostic biomarkers have been revealed previously. Genetically, genome-wide association studies (GWAS) have revealed that genes on chromosome 8q24, particularly the
PSCA gene (Prostate Stem Cell Antigen), were associated with increased metastatic potential of bladder cancer [
5,
6]. A hypothesis reasons that these genes detected by GWAS may be associated with androgen receptor responsiveness and inducing androgen-independent pathways, which stimulates tumor growth [
5]. The losses of regions on 10q (including
PTEN), 16q, and 22q, and gains on 10p, 11q, 12p, 19p, and 19q were positively associated with metastasis in muscle-invasive bladder cancers [
7]. With the genome-wide gene expression data, several studies have identified a combination of gene signatures to predict the prognosis of MIBC. Specifically, four gene signatures,
IL1B,
S100A8,
S100A9 and
EGFR, have been reported to have the capability of predicting MIBC progression [
8]. The novel combination markers of
USP18 and
DGCR2 can also predict survival in patients with muscle invasive bladder cancer [
9]. In addition,
NR1H3 expression is identified as a prognostic factor of overall survival for patients with muscle-invasive bladder cancer [
10]. However, there are some limitations for these studies. First, the gene signatures identified by these studies were not robust due to lack of validation dataset or small sample size in validation dataset. Second, comparative analysis was not conducted on the performance of these gene signatures for MIBC prognostic prediction. Third, the potential mechanism resulting in the worse prognosis has not been thoroughly investigated. In addition, the potential therapeutics for patients with worse prognosis was not proposed by these studies. In the present study, to avoid these limitations, we attempted to detect a combination of gene signatures for MIBC prognostic prediction and stratification. Based on the prognostic stratification, we also investigated the underlying molecular mechanism and potential therapeutic targets associated with worse prognosis of high-risk MIBC, which could improve our understanding of MIBC progression and provide new therapeutic approaches for these high-risk patients.
Discussion
Bladder cancer is one of the most common malignancies worldwide [
1]. Several studies [
8‐
10] have proposed several approaches to select and combine gene signatures for predicting the prognosis of MIBC, however, these gene signature sets have not been systematically compared with one another, and their performance on independent datasets are not satisfying. In the present study, we aimed to detect a combination of gene signatures for MIBC prognostic prediction and risk stratification. Based on the systematic data analysis, we identified three prognostic gene signatures,
KLK6,
TNS1, and
TRIM56, as the best subset of genes.
KLK6, a member of the kallikrein, was able to predict tumor recurrence in epithelial ovarian carcinoma [
26]. Moreover,
KLK6 has been reported to regulate epithelial-to-mesenchymal transition (EMT) and serve as prognostic biomarker for head and neck squamous cell carcinoma patients [
27], which also indicated that the poor prognosis in MIBC samples with high expression of
KLK6 might be associated with the dysfunction of EMT.
TNS1 was rarely reported to be associated with cancer, but was identified as a potential biomarker in human colorectal cancer [
23] and a regulator of metastatic potential in colorectal cancer via altering expression of genes involved in cell motility [
24]. In contrast, previous studies [
28,
29] have identified
TRIM56 as a tumor suppressor through activation of TLR3/TRIF signaling pathway, which was consistent with the result that
TRIM56 expression was a favorable indicator of MIBC in this study. Utilizing the expression profiles of these three signatures, we successfully built a multivariable Cox regression model to calculate risk scores and stratified the MIBC patients into high and low risk groups.
To demonstrate the high performance of the prognostic stratification based on MIBC risk prediction, we selected two independent cohorts as validation datasets. Remarkably, the stratified groups in the two validation datasets both exhibited significant difference in overall survival (Fig.
2,
P < 0.005). To further demonstrate the capability of the three-gene-signature in MIBC risk stratification, we also compared our three-gene-signature-based method with three other stratification methods by Wu et al. [
10], Kim et al. [
8,
9], and found that our method was superior to the others as patients stratified with our method exhibited a more significant difference in overall survival between high- and low-risk groups, suggesting that this prognostic stratification for MIBC was more robust and accurate. In addition, we also investigated whether this stratification was independent from other clinical indicators, such as lymph node and distant metastasis, and a history of radiation treatment, which could affect the MIBC prognosis. Consistently, the high-risk group exhibited worse prognosis than low-risk group in samples with and without lymph node metastasis, distant metastasis, and a history of radiation treatment. Specifically, we found that none of the three other stratifications selected the gene signatures based on univariable Cox analysis and their functionality. However, the present study selected the three gene signatures by integrating the univariable Cox analysis and Maximum Minimum Parents and Children (MMPC) algorithm, the strength of which is the maintenance of the statistical significance in both univariable and multivariable analyses, not only in univariable analysis.
Moreover, PI3K-Akt signaling pathway, a critical signaling pathway for cancer cell formation and progression [
30‐
33], was highly activated in the high-risk group according to the results from differential expression analysis and gene set enrichment analysis. In addition to PDGFRB, other upstream receptor tyrosine-kinases (RTKs) in PI3K-Akt signaling pathway, such as
EGFR,
CSF1R,
FGFR1,
FLT4,
FLT3,
NGFR,
NTRK1,
PDGFRA, and
TEK, were also observed to be upregulated in the high-risk group (P < 0.05, Additional file
3: Figure S1). These results further suggested that overexpression of these RTKs may be responsible for PI3K-Akt signaling pathway hyper-activation, and RTKs may serve as therapeutic targets in high-risk MIBC. Recently, an FGFR family inhibitor, erdafitinib, was approved by the U.S. Food and Drug Administration (FDA) to treat locally advanced or metastatic bladder cancer in adult patients with susceptible genetic alteration in
FGFR3 or
FGFR2, whose condition still progressed during or following prior platinum-containing chemotherapy. Therefore, we proposed that the erdafitinib treatment may work on patients of high-risk group, when platinum-containing chemotherapy failed to bring satisfying results.
In general, immune cells were infiltrated into tumor cells. We found that macrophage was highly filtrated into the high-risk MIBC (Fig.
7a, FDR < 0.05), and the angiogenesis-related genes were highly upregulated in high-risk MIBC (Fig.
7b, FDR < 0.05). More importantly, two M2 macrophage markers, CD163 and MRC1, were observed to be significantly upregulated in high-risk MIBC (Fig.
7c, P-value < 0.05). The co-occurrence of M2 macrophage infiltration and hyper-active angiogenesis in high-risk samples suggested that M2 macrophage may promote the angiogenesis of high-risk MIBC, which was consistent with previous studies [
34‐
36].
However, the present study still has some limitations. First, gene expression profiles of patients with long-term follow-ups should be collected to further assess the robustness of our stratification. Second, data regarding the efficacy of certain drugs in high-risk MIBC are not available, and in vitro and in vivo studies are needed to yield more experimental evidences. There is no experiment to validate the association between M2 macrophage and angiogenesis. Nevertherless, this study provides a new perspective on the molecular mechanisms behind high-risk MIBC, and has successfully illustrated how these mechanisms are related to the prognostic outcomes of MIBC patients.
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