Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most common malignancies in developed countries: there are 115,200 new cases and 49,000 death estimated for 2012 in Europe [1]; and the incidence of RCC is increasing in the USA, especially in young patients and high-grade disease [2]. The widespread use of abdominal ultrasonography for check-up or clarification of non-specific symptoms led to an increased detection of small-sized renal tumors. Small renal tumors are non-malignant in up to one third, but imaging does often not allow precise identification of non-malignant tumors, and thus potential harmful overtherapy occurs often. While the resection of small RCC is usually curative, the prognosis of metastatic RCC is poor: surgery (cytoreductive nephrectomy, metastasectomy [3]) and targeted therapy [4] improved patients survival, but eventually most patients succumb to the disease.

From a clinical point of view, a non-invasive biomarker could help to reduce overtherapy in case of small renal tumors, and furthermore, could be helpful for clinical monitoring during medical tumor therapy. Despite extensive research during the past years, there is still no biomarker for RCC. RNA microarrays are a powerful tool to identify dysregulated genes in cancer tissue. We therefore applied a microarray to identify RNAs dysregulated in RCC patients. Genes showing differential expression were then validated using qPCR and Western Blot.

In order to identify novel biomarkers for ccRCC, we investigated the mRNA expression profile in ccRCC using a microarray, and identified 1064 differentially expressed mRNA transcripts corresponding to 3 % of the investigated transcripts. As expected from earlier mRNA expression profiling studies in RCC patients, the expression profiling allowed a distinction of ccRCC and normal tissue accurately. The validity of the method was further confirmed by quantitative PCR experiments: 14 mRNA - so far not investigated by others - were quantified in a larger cohort (52 ccRCC and 51 normal renal tissues). Several other expression profiling [9‐12] studies also observed deregulation of these genes. Finally, we confirmed the dysregulation of selected genes on the protein level.

The dopamine transporter SLC6A3 (solute carrier family 6 member 3, also termed DAT1) regulates dopamine concentrations in the brain and expression changes are associated with Parkinson’s syndrome and attention-deficit/hyperactivity disorder. However, the function of this transmembrane protein in the kidney and in carcinogenesis is unknown. We observed a distinct increase of SLC6A3 mRNA (>200-fold) in ccRCC tissue compared to the normal parenchyma indicating a role as tumor suppressor gene. Surprisingly, protein levels were increased in the normal renal tissue as measured using Western Blot. It should be noted that approximately 40 % of the genes have differential protein/mRNA levels, most probably due to various levels of regulation during protein synthesis, e.g. posttranscriptional, translational, or posttranslational regulation [13, 14]. Notably, genes involved in mRNA processing pathways (release of introns, 3’end processing, mRNA export) are altered in ccRCC [15]. The more detailed histological analysis demonstrated that especially the proximal tubules expressed SLC6A3 at high levels, whereas its levels were moderate in the tumor. Interestingly, SLC6A3 expression was correlated with the time to progression, as shown in the Kaplan Meier diagram. Notably, the multivariate Cox regression analysis failed to confirm the prognostic value, but the small size of the cohort (n = 88) limited the statistical power. Of interest, high SLC6A3 mRNA levels were predictive for shorter overall survival within the TCGA cohort [7]. Notably, Seyednasrollah et al. suggest that solute carrier genes are highly expressed in ccRCC and have an impact on ccRCC patients outcome [16]. We also treated RCC cell lines with sertraline, an inhibitor of the SLC6A3 protein, and observed a dose-dependent, significant decrease of cell proliferation. It was earlier shown that sertraline induces apoptosis in HepG2 hepatoma cells as a result of the activation of the TNF-MAP4K4-JNK cascade pathway [17]. Thus, sertraline - often used as antidepressant and pain drug in palliative care settings in cancer patients - could be a potential therapeutic agent in metastatic ccRCC. It should be noted that sertraline, despite of being the most potent antidepressant at the dopamine transporter, also inhibits the serotonin and norepinephrine transporter [18]. The SLC6A3 protein can be visualized in SPECT and PET using radiolabeled ligands [19], it may be speculated that SLC6A3 could be used to identify metastases from ccRCC.

The relevance of NPTX2 (neuronal pentraxin 2) expression in ccRCC was reported during later stages of our experiments: van Roemeling et al. [20] also observed upregulation of NPTX2 primary tumors and metastases. Re-evaluation of the TCGA dataset by Seyednasrollah et al. also revealed NPTX2 (among many other genes) as predictive for poor outcome, but an internal validation cohort failed to reproduce the prognostic role of NPTX2 [16]. It was shown that cell viability and cell migration were promoted by interaction with the AMPA receptor resulting in an influx of calcium into cancer cells. As NPTX2 expression was functionally characterized earlier, we did not perform knockdown experiments. Notably, we did not notice a correlation of NPTX2 expression and clinical-pathological parameters, whereas van Roemeling et al. suggested an increase of NPTX2 in more advanced ccRCC [20]. In pancreatic cancer, NPTX2 expression is silenced by DNA hypermethylation [21].

Finally, we observed downregulation of FXYD4m KNG1 and SLC12A1 in ccRCC tissue. FXYD4 (FXYD containing ion transport regulator 4) modulates the Na-K-ATPase [22]. FYXD4 has not been implicated in human carcinogenesis so far. SLC12A1 (solute carrier family 12 member 1) is a kidney specific sodium-potassium-chloride co-transporter; it plays a key role in sodium chloride resorption. SLC12A1 downregulation was also noticed by Liu et al. in ccRCC, and its expression levels were negatively correlated with miR-21 expression [23]. A pathogenic role in cancer was further reported in ovarian cancer cell lines, which exhibited increased SLC12A1 expression levels [24]. KNG1 (kininogen 1) plays an important role in blood coagulation, but is also observed in renal tissue where it acts proinflammatory via the production of eNOS, NO, cGMP and prostacyclin [25]. Blockage of KNG1 binding to endothelial cells inhibited angiogenesis in a colon cancer mouse model [26]. Increased KNG1 levels were detected in sera of patients with hepatocellular carcinoma [27].

It should be noted that the composition of our study cohorts was probably unsuited to identify genes with prognostic impact: the microarray experiment included only one patient with metastatic ccRCC; locally advanced RCC (pT3) was observed in 60 % of patients but most patients had low grade ccRCC (G3 13 %). Also, the PCR and tissue microarray validation cohorts included only 13 % patients with distant metastasis. Thus, the power to identify genes with prognostic impact may be limited.

We have identified several novel biomarkers of potential diagnostic interest. The dopamine transporter SLC6A3 furthermore seems to be of prognostic interest, and could serve as therapeutic target in patients with metastatic ccRCC.