Inhibition of apoptosis is one of the basic pathogenetic steps implicated in carcinogenesis, enabling cells to prolong their survival, avoid immune surveillance, and resist cytotoxic therapies. Survivin, a member of the inhibitor of apoptosis protein family, is believed to play a role in progression of cancer and therapeutic resistance (Cohen et al., 2003).
In this work, all studied cases (100%) of CRC were immunopositive for Survivin. Other researchers reported figures of 79% (Chen et al., 2004), 78.5% (Jiang et al., 2004), and 100%. (Gianani et al., 2001).
Strong expression of Survivin has been reported in a range of solid malignancies including lung, stomach, breast, esophagus, colon, pancreas, urinary bladder, prostate, endometrium, uterine cervix, ovary, neuroblastoma, melanoma, and nonmelanomatous skin cancers. Expression has been reported in hematopoietic malignancies as well (Cohen et al., 2003).
Immunopositivity to Survivin varies according to the type of malignancy from 34% of gastric carcinomas to 93% of melanomas. Abd El Hameed (2005) explains this organ-dependent differential expression by intrinsic biological differences pertinent to the tumor studied and the type of antibody used (the higher affinity of polyclonal antibodies compared with monoclonal antibodies).
Whereas some authors claim that normal colonic mucosa does not express Survivin (Jiang et al., 2004; Abd El Hameed, 2005), others were able to detect Survivin mRNA transcripts (using RT-PCR) in histologically normal mucosa. Their conclusion was that Survivin expression in this setting might represent an ‘intermediate’ state marking the risk of neoplastic transformation (Sarela et al., 2000). Still, other researchers -ourselves included- were able to immunohistochemically detect Survivin in normal colonic mucosa. In this work, normal mucosae of four cases (9%) displayed uniform staining, a finding that agreed with that of Gianani et al. (2001).
In this work, as in others’ work (Kawasaki et al., 1998; Jiang et al., 2004; Abd El Hameed, 2005; Ponnelle et al., 2005; Li et al., 2007), no relation was found between Survivin expression on the one hand and patient’s sex, tumor grade, lymph node metastasis, or depth of invasion on the other. In the same context, Chen et al., 2004, working on the gene expression level, obtained the same result.
Conversely, other researchers (Lee et al., 2009; Na et al., 2009) found a statistically significant correlation between Survivin expression and lymph node metastasis and depth of tumor invasion. In their attempt to explain these contradictory reports, Brennan et al. (2008) claimed that this might stem from the varying specificity of the antibodies used or the inherent inter/intraobserver variability in interpreting Survivin immunostained sections.
In this study, like in others (Cohen et al., 2003; Lee et al., 2009), no relation was found between Survivin expression and length of disease-free survival; however, others could prove such a relation (Kawasaki et al., 1998). We did not establish correlations between Survivin expression and clinicopathological variables. This could be ascribed to the heterogeneity of studied cases vis-a-vis the clinical stage. In agreement with this, Adida et al. (1998) reported that the predictive value of Survivin expression was limited to patients with stage II CRC (Chen et al., 2004). Their finding was ‘later’ confirmed in Na et al.’s (2009) work, when they stated that ‘on stage wise analyses of patient survival, the Survivin expression was significantly correlated with overall survival and disease free survival, especially in stage II colorectal carcinomas.’
Analysis of the subcellular expression of Survivin is claimed by some authors to be of prognostic implication in stratifying patients with CRC. (Ponnelle et al., 2005; Stauber et al., 2007).
In this work, 95.5% of cases expressed Survivin in the cytoplasm, whereas only 4.5% showed nuclear expression. In line with our findings are those by Wang et al. (2010), who report that the subcellular localization was invariably in the cytoplasm. However, our figures strongly contrasted with those reported by Ponnelle et al. (2005), where 39% of colorectal tumors expressed Survivin in the nucleus and 41% in the cytoplasm.
In fact, according to Li et al. (2005), in their detailed review on the significance of subcellular localization of Survivin, among the 19 publications pertinent to Survivin subcellular localization in cancer tissues, nine proved nuclear expression as an unfavorable prognostic marker, whereas five proposed the reverse.
Several arguments have been put forward to explain these conflicting results. It has been proposed that procedural variations in immunostaining (Yin et al., 2006) and/or variable criteria used to label a tumor as nuclear Survivin or cytoplasmic Survivin positive might have played a role. (Yin et al., 2006; Qi et al., 2009) The difference in localization may also be ascribed to the tumor type and/or the biopsies examined, whether pretherapeutic or posttherapeutic (Qi et al., 2009). Li et al. (2005) provide another explanation relating to the presence of a number of splicing variants having different patterns of subcellular localization and hence variable biological roles. However, the currently available anti-Survivin antibodies recognize all the variants due to the existence of a common amino-terminal peptide. (Li et al., 2005).
Cohen et al. (2003) summarized the biochemical basis of subcellular localization in the amino-acid sequence of the carboxy-terminal domain of Survivin claimed to be responsible for its cytoplasmic localization and the nuclear localization of its splicing variant, Survivin _ Ex 3.
Distinct roles have been assigned to Survivin in each subcellular compartment. By maintaining the integrity of the mitotic spindle, nuclear Survivin displays its relation to the cell cycle (Cohen et al., 2003; Xie et al., 2006) being a prerequisite for cancer cell proliferation (Li et al., 2005; Stauber et al., 2007), whereas cytoplasmic localization of Survivin may promote its cytoprotective function by facilitating its interplay with the apoptotic machinery in cancer cells, thereby conferring an antiapoptotic effect (Stauber et al., 2007).
In the current work, no statistically significant relation was established between any pattern of subcellular localization of Survivin and length of progression-free survival (P=0.34). In the same context, Ponnelle et al. (2005) were able to prove that a high level of Survivin cytoplasmic expression was associated with better survival in patients with CRC, which contrasted with the findings of others (Cohen et al., 2003; Abd El Hameed, 2005), who demonstrated the reverse.
Acknowledging such a disparity in biological roles governed by variations in subcellular localization, some authors advocate quantifying not only the absolute expression levels of Survivin but also applying an immunoreactive score for cytoplasmic and nuclear forms (Stauber et al., 2007). Defining levels of nuclear versus cytoplasmic survivin might be of more prognostic relevance in CRC compared to measuring the total protein amount (Qi et al., 2009).
Unraveling the mechanisms of tumor cell invasion may aid the development of new treatment modalities that can hopefully arrest local invasion and metastatic spread of cancer. (Lee et al., 2009) In this context, research has long focused on elucidating the role of E-Cadherin, the prime mediator of epithelial intercellular adhesion (Debruyne et al., 1999).
Normal colonic mucosa shows a diffuse continuous ‘clean’ membrane immunopositivity along the lateral cell borders (Debruyne et al., 1999; Elzagheid et al., 2006)
The previous papers state the expected, logical loss of E-Cadherin expression in CRC (Dorudi et al., 1993; Khoursheed et al., 2003; Tsanou et al., 2008). Some authors attribute this loss primarily to gene silencing by hypermethylation of the promotor region (El Bahrawy et al., 2001). The loss of E-cadherin expression is associated with higher grade and more advanced stage in carcinomas of the breast, prostate, and urinary bladder (Khoursheed et al., 2003).
Over the years, different research groups have started to focus on a new theme, that is, the alteration in the subcellular localization of E-Cadherin from the membrane to the cytoplasm rather than an overall loss of staining (Aust et al., 2001; El Bahrawy et al., 2001; El Bahrawy et al., 2002; Guzińska Ustymowicz et al., 2004). Possibly, the ability to detect the marker in carcinomas may be related to technical advances in the sensitivity of the antibodies used. In accordance, cytoplasmic E-Cadherin was detected in 95.5% of our cases. Guzińska Ustymowicz et al. (2004) reported the same finding, claiming that the mere shift from membrane to cytoplasm leads to loss of function.
As an explanation for the cytoplasmic redistribution of E-Cadherin in colonic carcinoma, several possible mechanisms were proposed including downregulation of its tyrosine phosphorylation, (Nawrocki et al., 1998; Aust et al., 2001) increased E-Cadherin production rate, or failure to anchor to the membrane. The latter may be ascribed to alterations in a, ß, and y-catenins that link the E-Cadherin molecule to the actin cytoskeleton (Dorudi et al., 1993; Khoursheed et al., 2003). Other researchers add posttranslational modification as a possible factor governing changes in antigen availability (Dorudi et al., 1993; Elzagheid et al., 2006).
We report a statistical correlation (P=0.02) between the cytoplasmic expression of E- Cadherin and a longer disease-free survival in colon carcinoma cases studied. This contrasted with the work of Elzagheid et al. (2006), who claimed that ‘cytoplasmic expression more closely predicted disease recurrence and that reduction of membranous expression appeared to portend a poorer prognosis.’
This study found no significant correlation between E-Cadherin expression scores and the various clinicopathological variables, a finding that was reported previously (Khoursheed et al., 2003; Elzagheid et al., 2006). However, other researchers found that changes in E-Cadherin expression could correlate with a strong invasive potential (Tsanou et al., 2008).
In the current work, the expression of Survivin tended to correlate with that of E-Cadherin but statistical significance was not reached (P=0.06). We postulate that if a larger sample size is used, a positive relation might be established. Sarela et al. (2000) considered metastasis (and hence cell detachment from the surrounding matrix) and suppression of apoptosis to be parallel events. Detachment, according to Sarela et al., would trigger apoptosis in most adherent cells. Cells that express antiapoptosis genes (as Survivin) will be advantageous for survival and would continue to replicate.
In conclusion, Survivin was expressed in all cases of CRC studied. The expression of E-Cadherin does not decrease but shifts from membrane to cytoplasm. Neither Survivin nor E-Cadherin expression correlated with clinicopathological parameters. There is a significant correlation between the cytoplasmic expression of E-Cadherin (but not Survivin) and longer disease-free survival. The expression of Survivin tended to correlate with that of E-Cadherin.
Conflicts of interest
There are no conflicts of interest.
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