In parallel with the steady rise in ccRCC incidence over the past three decades, there continues to be considerable interest in identifying and exploring common molecular events that support the development and progression of ccRCC. In particular, much effort is currently focused on identifying molecular events within ccRCC tissues that correlate with well-known pathologic features of aggressiveness and patient outcome. Motivating these efforts is the understanding that identifying frequent molecular events within tumor tissue, especially those that correlate with known pathologic features of aggressiveness and disease progression, has the potential to provide insight into novel approaches to cancer prevention, prognosis and treatment. In the current study, we report that up-regulation of ET-2 in tumor tissue compared to patient-matched normal kidney samples is a common event in patients with ccRCC. In particular, we demonstrate that the up-regulation of ET-2 in ccRCC tumor tissue compared to adjacent normal tissue is more pronounced in tumors with less aggressive pathologic features. Moreover, we report that expression of ET-2 is also inversely associated with risk of ccRCC progression following surgery. That is, higher levels of ET-2 expression are associated with lower risk of development of distant metastasis after surgery. This inverse association is attenuated after adjustment for well known clinicopathologic features of ccRCC aggressiveness.
Of the three known ET peptides, to date only expression of ET-1 has been convincingly implicated in the development and progression of human cancer [15
]. With regard to ccRCC specifically, previous investigators have reported that ET-1 expression in both ccRCC cell lines [17
] and in human ccRCC tumors [18
]. Moreover, high expression levels of ET-1 have been reported to be associated with increased ccRCC aggressiveness, tumor progression and poor survival [19
]. By contrast, while over expression of ET-2 has been proposed as a novel mechanism by which human tumor cells can withstand hypoxic stress and support of tumor development and progression [7
], the data supporting this as a specific mechanism in ccRCC are very limited. Ohkubo et al [8
] were the first to report that human renal adenocarcinoma cell lines expressed high levels of the ET-2 mRNA transcript. Following that work several years later, Jiang et al [9
] used serial analysis of gene expression on renal adenocarcinoma cell lines to identify ET-2 as a gene that is inducible by hypoxia in the absence of a functioning von Hippel Lindau (VHL) gene. Given that loss of VHL is a common event in ccRCC [21
], these data suggest that up-regulation of ET-2 may represent a VHL-independent mechanism for ccRCC to respond to hypoxic stress and further support tumor growth. The importance of these cell line studies notwithstanding, we are aware of only one other investigative group that has directly examined the expression of ET-2 in human RCC tissue samples. In a small case series of 22 patients with surgically-resected ccRCC, Douglas et al [22
] observed no difference in mRNA expression levels of preproendothelin 2 (an intermediate precursor to ET-2) in tumor versus normal tissue; however, the small sample size and lack of centralized pathology review hampered the ability to draw meaningful conclusions or to explore evidence of differential expression across important subcategories.
Of interest, in our stratified analysis we report that over expression of ET-2 in ccRCC tumor compared to patient-matched normal kidney is more pronounced in those patients with tumors showing a less aggressive phenotype (i.e. low grade, early stage, no necrosis). One interpretation of our results is that over expression of ET-2 is an early event in the timeline of ccRCC carcinogenesis. To confirm our hypothesis, we mined in-house unpublished gene-expression microarray data where Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays were run on 15 primary RCC tumors and patient-matched lung metastases. Post frozen robust multiarray analysis normalization [23
], the average fold change of metastatic expression relative to patient-matched primary expression was 0.54 (p=0.007) for the probeset (206758_at) that mapped to ET-2. As such, therapeutics targeted at ET-2 could be studied further as a potential novel method for ccRCC chemoprevention (particularly in high risk individuals) as well as a neoadjuvant treatment for patients undergoing surgical excision of a clinically localized ccRCC tumor [24
]. Moreover, given that ET-2 encodes a secretory protein [5
], circulating levels of the ET-2 protein could also be explored as a marker of early disease in high risk populations as well.
The potential value of identifying molecular events that support ccRCC carcinogenesis notwithstanding, a key clinical issue in the field remains identifying biomarkers that correlate with ccRCC patient outcome (i.e. progression to metastasis after surgery). Related to this, the lethality associated with ccRCC results from tumors gaining the ability to invade surrounding tissues and metastasize to distant sites in the body. Indeed, five year survival drops from approximately 60% to less than 10% once a patient with clinically localized ccRCC progresses to distant metastatic disease [27
]. Motivated by this, there is a clear need to better understand the biologic underpinnings that support the mechanism of ccRCC metastasis and furthermore, to translate this understanding into improved patient care. The existing data indicating that ET-2 expression may mediate the development and growth of several human cancers, including ccRCC, suggests that this biomarker should be evaluated for its ability to predict ccRCC patient outcome following surgery. Moreover, our current understanding of the biologic function of ET-2 further suggests that higher expression levels of ET-2 would be predicted to be associated with less aggressive phenotype, lower risk of disease progression after surgery and improved survival. To date, however, investigations that have evaluated expression levels of ET-2 in human ccRCC tissue samples and their association with patient outcome are nonexistent. In our study, we are the first to report that higher expression levels of ET-2, a known mediator of cancer cell growth and proliferation, are associated with a lower risk of disease progression among patients undergoing surgery for clinically localized ccRCC. After adjustment for clinicopathologic predictors of ccRCC outcome, the association of ET-2 expression and PFS we observed was attenuated. While this diminishes the enthusiasm for ET-2 expression as a clinically relevant biomarker that could improve outcome prediction and enhance patient management it does not lessen the interested in ET-2 expression and its putative role in ccRCC progression. The prospect of utilizing ET-2 expression in the future to predict response to emerging therapeutics remains plausible. Moreover, if supportive data from mechanistic studies can be generated, our results do not preclude the possibility of developing adjuvant therapies that would enhance ET-2 expression signaling pathways to reduce the risk of progression in high risk patients. As such, these data represent an important advancement in our understanding of the potential role of ET-2 as a mediator of ccRCC progression.