Immunohistochemical staining of TMA
The tissue microarray technique was applied in this study. The total number of spots performed was 104; 100 spots represented the 50 studied cases of RCC (two spots per case) and four spots of normal kidney represented the control spots. A total of 96 tissue spots were informative for immunohistochemistry analysis including 61 CCRCC, 20 PRCC, five chRCC, two CDRCC, four SRCC, and four normal kidney tissue.
Expression of p21
Immunostaining showed that p21 was localized in the cytoplasm of normal renal tubular epithelium (Fig. 1a), whereas in tumor cells, immunoreactivity was most often nuclear (Fig. 1b–f). Thirty-six out of the 50 studied cases (72%) expressed p21. Analysis of the RCC tissue microarray for nuclear p21 expression showed the following distribution: negative score 0, 14 cases (28%); score 1, 20 cases (40%); score 2, nine cases (18%); and score 3, seven cases (14%).
p21 nuclear expression was found to increase with increasing tumor stage and this relation was statistically significant (P=0.022) (Table 1). In addition, a significant relation was found between p21 nuclear expression and the disease outcome of patients (P=0.050) (Table 2).
p21 expression scores were higher in metastatic RCC than in nonmetastatic RCC, where 60% of metastatic cases had moderate/high scores (scores 2 and 3) versus 20% of nonmetastatic cases. p21 expression scores were also higher in RCC with venous invasion than in RCC without venous invasion, where 85.72% of RCC with venous invasion had moderate/high scores (scores 2 and 3) versus 23.25% of cases without venous invasion, but these findings were not statistically significant (P=0.330 and 0.414, respectively). No significant correlation was found between p21 expression and each of the tumor histological subtype, tumor grade, tumor size, patient age, and patient sex (P=0.264, 0.543, 0.089, 0.123, and 0.981, respectively) (Table 1).
Expression of PTEN
In normal renal tubular epithelium, PTEN immunostaining was always cytoplasmic (Fig. 2a). In tumor cells, immunoreactivity was most often cytoplasmic. Nuclear reactivity was also observed but less often (Fig. 2b–f). Forty out of the 50 studied cases (80%) expressed PTEN. Analysis of the RCC tissue microarray for cytoplasmic and nuclear PTEN expression indicated the following distribution: negative score 0, 10 cases (20%); score 1, 19 cases (38%); score 2, 12 cases (24%); and score 3, nine cases (18%).
The PTEN expression score was significantly higher in PRCC than in CCRCC (P=0.003). In addition, PTEN expression was significantly higher in men than in women (P=0.017) (Table 1).
PTEN expression scores were lower in metastatic RCC than in nonmetastatic RCC, where 66.66% of metastatic cases showed a negative/low expression (scores 0 and 1) versus 54.29% of nonmetastatic cases. PTEN expression scores were also lower in RCC with venous invasion than in RCC without venous invasion, where 71.43% of RCC with venous invasion showed a negative/low expression (scores 0 and 1) versus 55.81% of cases without venous invasion. However, these findings were not significant (P=0.127 and 0.197, respectively). No significant correlation was found between PTEN expression and each of tumor size, tumor stage, and tumor grade as well as patient age and the disease outcome of patients (P=0.116, 0.675, 0.211, 0.226, and 0.530, respectively) (Tables 1 and 2).
Relation between p21 and PTEN expression
Out of the 34 cases with a negative/low p21 expression (scores 0 and 1), 17 cases (50%) showed a negative/low expression of PTEN (scores 0 and 1) and 17 cases (50%) showed a moderate/high expression of PTEN (scores 2 and 3). Out of the 16 cases with a moderate/high p21 expression (scores 2 and 3), 12 cases (75%) showed a negative/low expression of PTEN (scores 0 and 1) and four cases (25%) showed a high expression of PTEN (scores 2 and 3). The relation between p21 and PTEN expression was not statistically significant (P=0.095) (Table 3).
RCC is characterized by resistance to DNA-damaging chemotherapy. The mechanism of this effect, although incompletely understood, appears to be related to activation of survival pathways that occurs in the setting of repair of DNA damage (Lin et al., 2007).
p21 is a cyclin kinase inhibitor that is induced by p53 in situations of DNA damage. It likely plays a role in the decision pathways leading to apoptosis or DNA repair (Weiss, 2003). p21 has been shown to have multiple and diverse effects in various cancer-derived and noncancer-derived cell lines. Such effects include cell cycle promotion through cyclin/cyclin-dependent kinase assembly, positive effect on cell proliferation, and, more recently, its pleotropic functions in apoptosis (Park et al., 2008).
In the present work, immunohistochemical analysis of p21 was carried out in 50 cases of RCC including different histological subtypes and in normal kidney tissue. In tumor cells, immunoreactivity was most often nuclear. Cytoplasmic staining was encountered less often. In normal renal tubular epithelium, p21 staining was cytoplasmic. Interestingly, the subcellular localization of p21 may determine the differences in the function of this protein; cytoplasmic p21 has been shown to be pro-proliferative in vascular smooth muscle cells (Dong et al., 2004), whereas proliferating cell nuclear antigen binding and cyclin/cyclin-dependent kinase interaction occur when p21 is in its usual intranuclear location (Rössig et al., 2001).
In the present study, the relation between p21 nuclear immunostaining and the histological subtype of RCC was not statistically significant. It has been reported previously that nuclear p21 expression was the highest in CDRCC compared with all other RCC subtypes (Lam et al., 2007). This finding could not be assessed in the current work, because only one case of CDRCC was included.
In the present work, a statistically significant relation was found between p21 expression and the stage of RCC, where higher p21 nuclear expression scores were detected in more advanced stages of RCC. This finding is in agreement with the findings reported by Weiss et al. (2007), who reported that immunohistochemical expression of p21 correlates with more aggressive disease. Moreover, Perroud et al. (2006) reported that elevated levels of p21 predict a poorer prognosis in patients who have metastatic CCRCC. Contradictory results have been reported on the prognostic value of p21 in RCC. Samlowski (2009) reported that high p21 expression was associated with better prognosis in patients with localized RCC, whereas it was an indicator of poor prognosis in patients with metastatic RCC. Meanwhile, Haitel et al. (2001) found no prognostic value for p21 expression in RCC.
The present study also showed that higher p21 expression (scores 2 and 3) was more frequent in metastatic RCC and in RCC with venous invasion in comparison with nonmetastatic RCC and RCC without venous invasion. Although not reaching statistical significance, these findings suggest that p21 expression is associated with more aggressive disease.
A statistically significant relation was found, in the current work, between p21 expression and patient outcome, where moderate/high scores of p21 nuclear immunostaining (scores 2 and 3) were found in 75% of cases who died of their disease as compared with 35.48% of cases with no evidence of disease. These results suggest that p21 may have a prognostic value in RCC. Such findings are in agreement with the findings reported by Lam et al. (2007), who suggested that p21 may be a useful prognostic marker for RCC and may help in selection of patients for specific therapeutic approaches.
In the present study, the relation between p21 nuclear immunostaining, on the one hand, and tumor size, age, and sex of RCC patients, on the other, was statistically insignificant. In addition, the relation between p21 nuclear expression and RCC grade was not significant. Weiss et al. (2007) reported that p21 nuclear positivity correlated with tumor grade in CCRCC, but was only significantly increased in grade 3 compared with grade 1. The paradox that p21 nuclear expression correlated with tumor stage but not with tumor grade or size in the present study may be explained by the fact that nuclear grade has been reported to be an important predictor of survival in CCRCC but not in papillary, chromophobe, or SRCC (Medeiros et al., 1997; De Peralta-Venturina et al., 2001; Minervini et al., 2002), which altogether represent 35% of our cases. In addition, 50% of our cases were stages III and IV, in which other factors (other than tumor size), namely, tumor extension beyond the kidney, lymph node metastasis, and distant metastasis contribute toward tumor stage.
It is well known that inactivation of apoptosis is essential for cancer development. Thus, a major strategy for chemotherapy involves channeling malignant cells into apoptotic pathways, and, consequently, a frequent cause of failure of chemotherapy results from malfunction of apoptosis. As p21 is increased in DNA damaged cells, likely in an attempt to repair the damage, its antiapoptotic property can be used to therapeutic advantage in chemotherapy-resistant cancer. Transient attenuation of this protein in combination with conventional DNA-damaging chemotherapy would sensitize kidney cancer cells to apoptosis by directing cancerous cells into the apoptotic, rather than the DNA repair pathway (Park et al., 2008). It has been suggested that p21 plays a key role in the mechanisms by which tumors deficient in PTEN resist chemotherapy (Lin et al., 2007).
PTEN is a ubiquitous regulator of the cellular PI3k signaling pathway. This pathway is characterized by the regulated activation of class I PI 3-kinase enzymes, producing phosphatidylinositol 3,4,5-trisphosphate (PIP3), which, in turn, mediates downstream signaling through a range of effector proteins, including the proto-oncogene product protein kinase B (PKB)/Akt. PTEN antagonizes PI3k signaling by dephosphorylating PIP3, thus inactivating downstream signaling and inhibiting cellular proliferation, survival, and growth (Leslie and Downes, 2004). The PTEN tumor suppressor gene is frequently mutated or deleted in a wide variety of cancers and these tumors are generally more aggressive and resistant to treatment than those with the wild-type PTEN (Lin et al., 2007). RCC is commonly associated with treatment failures (∼90% in metastatic cases), and these tumors frequently have PTEN abnormalities (Youssif et al., 2011).
In the present work, TMA sections representing the 50 studied cases of RCC were stained with the anti-PTEN antibody and compared with normal kidney control. The normal kidney showed diffuse strong cytoplasmic staining of tubular epithelial cells. In RCCs, PTEN staining was predominantly cytoplasmic in the majority of cases (only 10% of the cases showed predominantly nuclear staining) and varied in different cases. These findings are in agreement with those of Pantuck et al. (2007), who reported that the anti-PTEN antibody stained the cytoplasm of RCCs and that tumors showed a lower expression than normal renal tissues. Similarly, Perren et al. (1999) and Zhou et al. (2002) described tumors with reduced PTEN expression relative to the surrounding tissues in their studies on colorectal cancer and breast cancer, respectively. Immunohistochemical studies of a variety of tissues have shown that the protein is largely cytoplasmic, although in some cases, predominantly nuclear staining was found (Perren et al., 1999; Tachibana et al., 2002; Zhou et al., 2002). It has been suggested that nuclear compartmentalization of PTEN may be related to its tumor-suppressive activity; however, its nuclear function remains poorly defined (Song et al., 2011).
In the current work, 58% of the studied cases of RCC showed a negative/low PTEN expression (scores 0 and 1). Similarly, Sitaram et al. (2009) reported that reduced PTEN protein expression is encountered in a high frequency in RCC. Meanwhile, Figlin et al. (2005) reported PTEN loss in only 20% of RCCs. PTEN protein and gene expression have been described variously as reduced (Hara et al., 2005), absent (Brenner et al., 2002), mutated (Alimov et al., 1999), or deleted (Sükösd et al., 2001) in human RCCs. Brenner et al. (2002) reported that loss of PTEN occurs during renal carcinogenesis. There is substantial evidence now that loss of PTEN expression is far more frequent than mutation of the PTEN gene in most cancers; thus, future research must focus on processes regulating the transcription of the PTEN gene and/or turnover of the PTEN protein (Leslie and Downes, 2004).
In the present study, PRCC showed significantly higher PTEN expression than CCRCC, suggesting that PRCC may less likely benefit from mammalian target of rapamycin (mTOR) inhibitor therapy than CCRCC. Furthermore, two out of the three studied cases of chRCC showed high PTEN expression. These findings are in agreement with those of Pantuck et al. (2007), who reported higher PTEN expression in nonclear cell subtypes of RCC, and thus proposed that not all RCC tumor types are equally amenable to mTOR-targeted therapy.
In the current work, most of stage IV cases and all the Fuhrman grade 4 cases showed negative/low PTEN expression (scores 0 and 1). In addition, negative/low PTEN expression (scores 0 and 1) was more frequently observed in metastatic RCC and in RCC with venous invasion in comparison with nonmetastatic RCC and RCC without venous invasion. Although not reaching statistical significance, these results predict that patients with lower PTEN expression do worse than those with higher PTEN expression. These findings are in agreement with those of Leslie and Downes (2004), who reported that PTEN loss is associated with adverse prognosis in RCC. In addition, Cho et al. (2007) reported that lack of PTEN expression is a negative prognostic factor for disease-specific survival in patients with metastatic RCC. The lack of statistical significance in our results may be attributed to the limited number of cases included.
On correlation of PTEN expression with patient outcome, negative/low expression (scores 0 and 1) was detected in 75% of patients who died of their disease, 60% of patients who were alive with disease, and 54.84% of patients with no evidence of disease. This is in agreement with results obtained by Kim et al. (2005), who reported that low PTEN expression correlates with worse survival. PTEN as well as pAkt, p27, and pS6 have been suggested to serve as surrogate parameters for predicting prognosis in RCC (Pantuck et al., 2007).
One of the important pathways related to cancer is the mTOR pathway, which involves downstream signaling from PI3K/Akt that leads to phosphorylation of mTOR, thus activating its substrate, p70S6 kinase, in turn promoting mRNA translation, cell cycle progression, and angiogenesis. This pathway is of special interest in RCC because of frequent inactivation of PTEN in these tumors (Youssif et al., 2011). In PTEN-deficient cells, increased PIP3 levels result in constitutive activation of PKB/Akt, which itself lies upstream of mTOR, with subsequent stimulation of growth-promoting pathways. Thus, the mTOR inhibitors may be ideally suitable for use in RCC (Weiss and Lin, 2006) and patients with a highly activated mTOR pathway are expected to benefit most from this therapy (Pantuck et al., 2007). The therapeutic benefit from mTOR inhibitors has been variable among RCC patients who were selected only with respect to patient-related clinical factors, without considering tumor-related factors (Youssif et al., 2011). Immunohistochemical detection of PTEN expression in RCC, as performed in the present work, may be helpful in selection of patients who can benefit from mTOR inhibitor therapy.
In the present study, p21 expression was correlated inversely with PTEN expression, but the statistically significant level was not reached, possibly because of the limited number of cases studied. The inverse correlation between p21 and PTEN expression has also been reported by Yoo et al. (2006), who detected higher levels of p21 protein and transcripts in PTEN-deficient bladder tissue. Chen et al. (2005) observed a similar phenomenon in PTEN-deficient prostate tissue, which they interpreted as confirming the activation of a p21-related senescence pathway. The inverse correlation between p21 and PTEN expression may be explained by the fact that PTEN inactivation renders it unable to carry out its phosphatase function. PIP3 can no longer be deactivated; thus, it continues to propagate its signal downstream, with continuous activation of PKB/Akt. p21 is phophorylated by Akt. This phophorylation increases p21 stability (Zhou et al., 2001). Thus, PTEN inactivation augments p21 stability.
Using a PTEN knockdown cell line, Lin et al. (2007) showed that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding that was associated with increased levels of p21. They concluded that augmentation of p21 may be an important mechanism through which PTEN-altered tumors acquire resistance to chemotherapy. They also reported that PTEN-deficient tumors are sensitive to mTOR inhibitors, which, in turn, sensitize tumors to DNA damage-induced apoptosis through inhibition of p21 translation.
Recent breakthroughs have led to greater knowledge of the molecular genetic and oncogenic pathways of RCC. This would allow for individualized application of systemic and targeted therapies on the basis of the molecular signature of the tumor to be targeted, thus ultimately improving patient outcome (Leppert et al., 2007). The findings of the present work suggest that p21 expression may serve as a poor prognostic indicator in RCC and that PRCC show higher PTEN expression, and thus may less likely benefit from mTOR inhibitor therapy as compared with CCRCC. Immunohistochemical detection of expression of p21 and PTEN may help in identification of the appropriate candidates for targeted therapy; however, larger studies on these two markers are required before such therapeutic modalities become established.
Conflicts of interest
There are no conflicts of interest.
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