Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma

1. Introduction

Since cancer stem cells (CSCs) were introduced in solid tumors in the early 2000s [1, 2], targeting of CSCs has become an important aim. Therefore, finding the molecular markers to isolate and characterize the CSCs among the tumor cells are urgently needed.

Renal cell carcinoma (RCC) accounts for 90% of adult renal malignancies and is the most lethal urological cancer with an annually increasing incidence [3, 4]. It is estimated that there will be 63,990 new cases of RCC in the United States and 14,400 deaths in 2017 [4]. RCC is divided into several histological subtypes. Clear cell as the most common subtype, comprises 70% of RCC cases, while papillary and chromophobe RCC are 10% to 15% and 5% of RCC cases, respectively [5].

CD105 (endoglin) is a transmembrane glycoprotein and the receptor for transforming growth factor (TGF). CD105 regulates TGF- β signaling by interacting with TGF- β receptors I and/or II. Several studies have shown that endoglin contributes to the development of blood vessels and angiogenesis and is essential for tumor growth and development of metastasis. In addition, CD105 is a prominent marker for mesenchymal stem cells (MSCs) [6, 7].

CD105 has been introduced as a potential prognostic marker in RCC. However, the results are inconclusive and conflicting [8, 9]. Recently, CD105 has been described as a CSC marker for RCC by Bussolati and her colleagues who first isolated a population of tumor-initiating cells by means of cell sorting using CD105 marker from patient s’ specimens after radical nephrectomy. These cells displayed tumor-initiating ability and could generate serially transplantable carcinomas. Moreover, CD105 + cells had much stronger features of CSCs compared with CD105 - cells. The CD105 + renal CSCs expressed mesenchymal stem cell markers such as CD44, CD90, CD146, CD73, CD29, embryonic stem cells markers such as Nanog, Oct4, Musashi, Nestin, and embryonic renal marker Pax-2 but lacked differentiated epithelial markers. Functionally, these cells were able to generate spheres in a non-adhesive culture system [10, 11]. In addition, CSCs have been identified and isolated using CD105 from the various RCC cell lines including 786-O, SMKTR2, SMKTR3, 769-P, Caki-1, Caki-2, ACHN, and RCC-6 [12]. A very recent study by Hu at al. on the CD105 + population in human RCC xenograft models demonstrated that xenograft tumor-derived CD105 + subpopulation displays stem-like characteristics with increased self-renewal compared to parental cells and also, CD105 + cells were more resistant to chemotherapeutic agents. They concluded that CD105 is not only a biomarker for renal CSCs but also confers self-renewal capacity and contributes to chemoresistance in RCC [13].

An immunohistochemical study, which was carried out on paraffin-embedded tumor samples derived from 102 ccRCC patients showed that expression of CD105 in the cytoplasm of tumor cells correlates with Fuhrman nuclear grade and tumor stage and tumoral CD105 expression is also an independent prognostic factor in these patients [14]. Limited information is available on the association of CD105 expression as a CSC marker, in the RCC patients and the clinicopathological characteristics.

RCC tends to be a highly vascular tumor and previous studies have reported the presence of microvascular invasion (MVI) in up to 45% of RCCs [15]. Microvascular invasion has been considered as a predictive factor of potential spread of tumor cells, and also has been identified as an independent risk factor for poor prognosis in many solid tumors such as RCC [16]. Since CSCs play an important role in tumor development and progression [17, 18], investigations on the possible association between expression of CD105 as a CSC marker and microvascular invasion (MVI) as well as lymph node invasion (LNI) is required. Therefore, we hypothesized that cumulative expression of CD105 and also the presence of MVI and LNI can predict the outcome in patients with RCC after radical nephrectomy. Additionally, given the importance of histopathologic subtype in the treatment planning and targeted therapy of RCC, further investigation is required to clarify the role of CD105 as an independent prognostic marker in different subtypes of RCC. Therefore, the current study was designed as the first attempt to investigate the expression and prognostic value of CD105 in a series of well-defined RCC samples including clear cell, type I and II papillary, and chromophobe RCCs using the tissue microarray (TMA) technique. Furthermore, the possible association between expression level of CD105 among various subtypes of RCC was examined.

3. Results

4. Discussion

CD105 is a tumor marker expressed in vascular endothelial cells and strongly correlates with processing of new blood vessel formation [6]. In addition, high tumor micro vessel density (MVD) as assessed by CD105 immunohistochemistry shown to be associated with poor prognosis in several solid tumor types [7]. Recent studies have shown that CD105 is also expressed in tumor epithelial cells of RCC [21], human hepatocellular carcinoma (HHC) [22], and ovarian cancer [23]. Recently, CD105 has been described as a renal CSC marker [10]. Bussolati et al. and Hsieh et al. showed that the cell surface marker CD105 which is also one of the mesenchymal stem cell markers can be the marker of renal cell carcinoma as well as sarcomatoid renal cell carcinoma [10, 24]. The results by Bussolati et al. indicated CD105 + were clonogenic, expressed several mesenchymal stem cell markers, embryonic stem cells markers and lacked differentiative markers, could differentiate in vitro into epithelial and endothelial cell types, and could generate in vivo serially transplantable tumors [10]. CD105 were also investigated to determine the prognostic value in RCC. In contrast, expression of CD105 in sections of formalin-fixed paraffin-embedded of RCC tumor samples was cytoplasmic. They concluded that CD105 was a predictor of patient’s outcomes than other clinicopathological factors [14]. In a new study by Hu at al. it has been shown that CD105 is essential for the maintenance of self-renewal and chemoresistance in renal CSCs [13]. According to the theory of CSCs, these subpopulations are responsible for driving tumor growth, progression, angiogenesis, resistance to chemotherapy agents, and radiotherapy [18]. A previous study on HCCs showed that CD90 + papulation share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105 [25]. These results suggested that CD105 is not only a vascular endothelial cell marker but also can be a marker of CSCs with mesenchymal cell features [22].

In the current study, we showed highly significant association between the median of the cytoplasmic CD105 expression and RCC subtype including a significant difference in the median of the expression level of CD105 between ccRCC and papillary RCC cases (Type I and II) and also ccRCC and chromophobe RCC ( 0.001). In addition, a significant association was found between cytoplasmic and endothelial CD105 expression and RCC subtypes. Moreover, by analyzing the three subtype of RCC, we found that ccRCC and papillary RCC would have a higher level of tumoral cytoplasmic CD105 expression compared to chromophobe RCC. It might be due to their origin which ccRCC and papillary RCC are derived from the proximal epithelial tubule cells, while chromophobe RCC is derived from a distal epithelial tubule cells [26].

In this study, we showed that tumoral cytoplasmic and endothelial expression of CD105 was significantly associated with advanced pathological stage in ccRCC. Importantly, we observed that the median of the tumoral cytoplasmic CD105 expression was higher in advanced stages (stage III and stage IV) in comparison with lower stages (stage I and stage II), which shows the association of cytoplasmic CD105 expression with tumor aggressiveness in ccRCC. In addition, we found that the tumor stage and Fuhrman nuclear grade were independent prognostic predictors for OS in the multivariate analysis in both tumoral cytoplasmic and endothelial CD105 expression. Our results regarding endothelial CD105 expression are consistent with those of previous studies in ccRCC cases [8, 14, 27]. It is shown that vascular endothelial CD105 expression strongly correlates with angiogenesis [7] and also CSCs are associated with angiogenesis during the growth and development of tumors [28]. The study of Saroufim et al. [14] investigating cytoplasmic CD105 expression in renal cancer, the tumor stage was found to be a significant predictor of the OS in the univariate analysis but not in the multivariate analysis. Our different results may be due to a larger number of subjects compared to the aforementioned study. The tumor stage and nuclear grade are the most reliable prognostic factors in RCC and tumors with a high Fuhrman nuclear grade have a more aggressive phenotype and also are associated with local invasion and distant metastasis [29]. Furthermore, we showed for the first time, a significant association between endothelial CD105 expression and tumor size. Also, tumor size was found to be a prognostic indicator in univariate analysis, in contrast with a previous study which could not find any significant association between endothelial CD105 expression and tumor size [9]. Investigations have shown that tumor size has important clinical and pathologic implications in RCC patients and is significantly associated with risk of metastasis [30, 31].

In our study, an association was found between higher level of tumor cytoplasmic and endothelial CD105 expression and MVI as well as renal vein in ccRCC which have very important clinical indications. It is shown that RCC patients demonstrating MVI or a high degree of angiogenesis are at high risk for developing metastatic disease [32], therefore, MVI is related to cancer progression and survival in RCC and has a more significant on prognosis compared to the macroscopic renal vein or vena cava invasion following surgical tumor resection [33, 34]. The results of survival analysis showed a statistical significant relationship between patients with MVI and worse OS and higher stage. It has been reported that tumor vasculature may have a role in supporting and maintaining cancer stem cells [17, 35].

LNI is also one of the other most important prognostic factors in RCC. A previous study showed that LNI provided the strongest prognostic information for patients without MVI after radical nephrectomy [36]. In our study, we did not find any association between cytoplasmic expression of CD105 and LNI as well as survival outcomes.

In addition, significant association was found between the level of tumor cytoplasmic and endothelial CD105 expression and renal sinus involvement showing that CD105 protein expression is related to the degree of malignancy in ccRCC [37].

In this study, overall survival rate of ccRCC patients with high tumoral cytoplasmic and endothelial CD105 expression was significantly lower compared to the patients with low CD105 expression ( P = 0.032, P = 0.004, respectively) in univariate analysis. In addition, ccRCC patients who expressed higher level of CD105 in tumor cytoplasm and endothelial blood vessels had shorter 5-year overall survival compared with those with low expression. However, the pattern of CD105 expression was not a significant predictor of survival in multivariate analysis; i.e. CD105 expression was not found to be an independent prognostic factor for predicting ccRCC patient’s OS. That is likely due to low numbers of events therefore, a lengthier follow up is still needed. A previous study concluded that CD105 is an independent prognostic marker in these patients [14]. Therefore, we determined that higher levels of tumor cytoplasmic and endothelial CD105 expression was associated with increased tumor aggressiveness, more advanced disease, and poor prognosis in ccRCC. It is proposed that these clinical findings are related to the characteristics of cancer stem cells, as increased numbers of CSCs can contribute to an aggressive tumor behavior in various cancers and is significantly associated with metastatic potential [18, 38]. In addition, studies showed that CSC markers are associated with tumor progression in many solid tumors [39].

Additionally, we did not find any association betw- een cytoplasmic and endothelial expression of CD105 and various clinicopathological features or survival data in the examined type I and II papillary and chromophobe RCC which are less frequent than the ccRCC subtype. Our result is in agreement with a recent study by Matak et al. who demonstrated that CD105/CD133 cannot be validated as cancer stem cell markers in papillary RCC cell lines [40]. Our study, is the first report showing cytoplasmic expression of CD105 in chromophobe RCC.

Several previous studies have shown that anti-CD105/endoglin monoclonal antibody can effectively reduce or suppress angiogenesis, tumor growth and metastasis in SCID mice [41]. TRC105, a novel chim- eric IgG1 monoclonal antibody that binds CD105, currently is being evaluated in phase-Ib clinical studies in patients with advanced solid tumors. Ongoing clinical trials are testing TRC105 in combination with bevacizumab in glioblastoma, and together with VEGFR TKIs in RCC, hepatocellular carcinoma, and soft tissue sarcomas [42]. Results of the current study showing the differential expression of CD105 in the tumor cytoplasm and endothelial vessels of various subtypes of RCC provides important information regarding the etiology of tumor aggressiveness and prognosis in these tumors which may help in finding new therapeutic strategies for various subtypes of RCC possibly using CD105 as a therapeutic target.

5. Conclusion

In summary, our results revealed that there is a statistically significant difference between tumor cytoplasmic and endothelial CD105 expression in various subtypes of RCC. We showed that tumor stage and nuclear grade were independent prognostic variables for OS in ccRCC patients. In addition, higher level of tumor cytoplasmic and endothelial CD105 expression was associated with MVI and MVI correlated with advanced stage. Moreover, we found that higher expression levels of tumor cytoplasmic and endothelial CD105 were associated with more aggressive behavior, tumor progression, and poor OS in ccRCC subtype. However, such associations were not found in type I and II papillary and chromophobe RCC. Therefore, we consider the CD105 CSC marker as a useful prognostic molecular marker and potentially a target molecule for targeted therapy only in ccRCC, but possibly not in other subtypes of RCC.