Evaluation of myosin VI, E-cadherin and beta-catenin immunostaining in renal cell carcinoma

Renal cell carcinoma (RCC) is a cancer of increasing incidence and mortality [1]. By diagnosis, up to one-third of patients already have a metastasised disease and half of the remaining patients will suffer a recurrence after curative treatment [2]. The behaviour of RCC can be difficult to predict even though there are many well-known prognostic factors for the disease. Myosins are a large family of molecular motor proteins and their immunoexpression has previously been demonstrated in variety of epithelial cancers including RCCs [3–5]. Myosin VI is one of the so-called unconventional myosins that moves in a reverse direction when compared to the other known myosins, i.e. it moves from the plasma membrane into the cell and away from the surface of internal organelles such as the Golgi complex. Myosin VI plays a role both in transporting and anchoring the cells and takes part in a wide range of cellular processes such as endocytosis, exocytosis, cell migration, cell division and cytokinesis [3, 6]. Furthermore, myosin VI is linked to E-cadherin and beta-catenin in ovarian cancer [7].

One of the key processes in developing metastasised disease is a loss of cellular adhesion [8]. E-cadherin, a member of the adhesion molecule family of cadherins, mediates predominantly cell-cell adhesion in epithelium and epithelial tumours. It is a tumour suppressor, the loss of which is known to worsen the prognosis of many cancers. The whole function of E-cadherin is not yet well understood but it might influence the transcription of target genes [9–15]. Transmembranic glycoprotein E-cadherin interacts with the cytoskeleton via intracellular proteins named catenins. Cell-cell cohesion can be damaged by the loss of E-cadherin expression or changes in catenin expression, which leads to the loss of cadherin function. The cadherin-catenin complex also influences migration and modifies cell growth and the survival of neoplastic cells [8]. In addition, beta-catenin, a member of the catenin family, participates in signal transduction [16, 17].

There are no current immunohistochemical prognostic markers for RCCs in routine use. In this era of new treatment possibilities there remains a need for better prognostic tools to plan the treatment and follow-up of RCC patients. The purpose of this study was to examine for the first time the immunostaining of myosin VI in RCCs and to investigate the prognostic potential of immunostaining myosin VI, E-cadherin and beta-catenin in RCCs.

This was the first study characterising the expression of myosin VI in RCCs. Here, cytoplasmic myosin VI immunopositivity was associated with the lower Fuhrman grades of RCCs, but in multivariate Cox regression model it was also a marker of poorer prognosis. The immunoexpression of myosin VI has been demonstrated in prostatic adenocarcinoma [21, 22]. There is also evidence that links myosin VI to the migration of human ovarian cancer cell lines [23]. In ovarian carcinomas, myosin VI expression has been associated with the aggressive behaviour of the tumour [24]. In our study, cytoplasmic myosin VI immunostaining was not a statistically significant prognostic factor according to log rank test. However, in multivariate Cox regression model adjusted with the known prognostic factors of RCCs, stage and Fuhrman grade, cytoplasmic myosin VI immunostaining was a prognostic marker for RCC specific survival. This means, that confounding factors affecting the results of log rank test were present, which could be reduced in Cox regression model. Noteworthy, the HR for cytoplasmic myosin VI immunostaining was increased also when tumour diameter, age or gender was retained to the model. Despite the association of cytoplasmic myosin VI immunopositivity with lower Fuhrman grades, the mean survival times for subjects with cytoplasmic myosin VI immunopositive Fuhrman grade II tumours died of RCC during follow-up were shorter in comparison with subjects with cytoplasmic myosin VI immunonegative tumours. So cytoplasmic myosin VI immunopositivity seems to have prognostic potential also within Fuhrman grade II tumours but not only within poorly differentiated tumours.

It has been reported that membranous beta-catenin immunoexpression is downregulated in conventional RCCs with low nuclear grades but higher in papillary and chromophobic carcinomas than conventional RCCs [25]. In our study, nuclear beta-catenin immunostaining was more frequently detected in cases with lower Fuhrman grades, but we found no prognostic significance of beta-catenin immunostaining in RCCs. Furthermore, we detected no differences in beta-catenin immunoexpression patterns between the different histological subtypes of RCCs.

According to our study, nuclear E-cadherin expression is neither an independent prognostic factor in RCC-specific survival nor associated with the nuclear grade of the tumour. Nuclear E-cadherin has previously been demonstrated to be associated with better prognosis of RCCs [15], and there has also been a reported downregulation of E-cadherin expression in clear cell RCCs [26]. In our study population, we could not prove the prognostic importance of E-cadherin that had previously been shown in smaller study populations and with shorter follow-up times. In previous studies, nuclear E-cadherin expression was detected only in clear cell RCCs [15]. In our study, some nuclear positivity was also demonstrated in papillary and chromophobic carcinomas.

According to our study, nuclear myosin VI is associated with beta-catenin but there is no relationship between myosin and E-cadherin in RCCs. Myosin VI is linked to E-cadherin and beta-catenin and participates in border cell migration where it stabilises the E-cadherin-beta-catenin cell adhesion complex [7]. Myosin VI is a cytoplasmic protein and the significance of nuclear myosin VI immunostaining is unknown. Beta-catenin, however, can be detected in the nucleus in various carcinomas [27–30]. Nuclear myosin VI could be a regulating factor for beta-catenin or a co-worker. The association between myosin VI and beta-catenin might also suggest that beta-catenin provides a molecular mechanism for signal transduction from the cytoplasm to the nucleus of the cell, thereby also influencing myosin VI gene expression. Beta-catenin plays a role in the Wnt (wingless type) pathway where the multiprotein destruction complex which involves APC (adenomatous polyposis coli) influences the phosphorylation and unphosphorylation of beta-catenin and has been demonstrated to lead to the transcription and expression of oncogenes such as c-myc and c-jun [16, 17]. Beta-catenin has also been reported to be capable of regulating gene expression by the direct interaction with transcription factors such as LEF-1 (lymphoid enhancer-binding factor), providing a molecular mechanism for a signal transmission from cell-adhesion components to the nucleus [16]. In our study, we also found a positive relationship between nuclear E-cadherin and beta-catenin immunostaining, which has previously been detected in gastrointestinal carcinomas [15, 31, 32].

Our findings were that in RCCs there is immunoexpression of myosin VI in cytoplasm and nucleus, and cytoplasmic myosin VI is an independent prognostic factor in RCC-specific survival. In the future, myosin VI may have use as a prognostic marker of RCCs. Cytoplasmic myosin VI immunopositivity and nuclear beta-catenin immunostaining were associated with lower Fuhrman grades but not stages. Nuclear myosin VI and beta-catenin immunoexpression are associated with each other. Nuclear E-cadherin and beta-catenin immunostaining patterns are also positively related together. The discrepancy with previous studies concerning the prognostic importance of nuclear E-cadherin in RCCs might be because of different study populations and follow-up times.