Overexpression and hyperphosphorylation of retinoblastoma protein in the progression of malignant melanoma

Abstract

Mutation, absence or abnormal functioning of retinoblastoma protein are fundamental elements of uncontrolled growth in human cancer. In this study, we analyze the expression of retinoblastoma protein and phosphorylated retinoblastoma protein in melanocytic tumors in vivo. Real-time RT-PCR and immunohistochemistry (tissue microarrays and conventional histological sections) reveal that retinoblastoma protein is progressively upregulated in advanced and metastatic malignant melanomas in vivo. However, this increase is paralleled by increased retinoblastoma protein inactivation due to protein phosphorylation. Interestingly, retinoblastoma protein phosphorylation occurs not homogeneously, but with a 'growth zone'-related pattern. In superficial spreading melanomas a subepidermal-lateral maximum of phosphorylated retinoblastoma protein can be frequently observed. Accordingly, nodular vertically invasive melanomas are characterized by a strong staining of phosphorylated retinoblastoma protein in deep-dermal invading protrusions of the tumor. Furthermore, Kaplan-Meier analysis of 13 cases of advanced melanomas with long-time follow-up suggests a significant negative impact of retinoblastoma protein phosphorylation on survival of melanoma patients independent of tumor thickness. We conclude that the evaluation of phosphorylated retinoblastoma protein in melanocytic tumors could become a helpful adjunct in clinicopathological routine. The main principle of cell cycle homeostasis is the phosphorylation-dependent activation and deactivation of regulatory proteins upstream of retinoblastoma protein (pRb), which represents the central cycle-controlling element. At the G1/S transition, the antiproliferative function of active, hypophosphorylated pRb is mediated by its binding capacity to proproliferative transcription factors such as E2F, c-Myc, ATF-2 and c-Abl. 1 Surprisingly, with the exception of retinoblastoma, osteosarcoma and small cell lung carcinoma, the overall rate of pRbmutations in the vast majority of human cancers is either extremely low or not existent. 2 Also in malignant melanomas, the loss of cell cycle control is thought to be due to a lack of pRb activity and not to lack of expression or mutation. 3,4 Currently, the concept is arguably accepted that persistent inactivation and hyperphosphorylation of wild-type pRb in melanomas is caused by a sustained cyclindependent kinase (cdk) activity. [5][6][7] In uveal malignant melanomas, Brantley et al 8,9 have recently reported an increased pRb phosphorylation at Ser807/811. Since they have also detected a high cyclin D expression, the hypothesis of a functional inactivation of pRb due to cyclin-and cdk-dependent hyperphosphorylation was further substantiated.To the best of our knowledge, no detailed studies have been performed to elucidate the pRb phosphorylation status in in vivo samples of cutaneous melanocytic tumors. In particular, studies on the impact of hyperphosphorylated pRb on melanoma progression are still lacking. Therefore, in this study we analyze to...

Loss of dipeptidyl peptidase IV immunostaining discriminates malignant melanomas from deep penetrating nevi

“…Consistent with our previous findings on the progressive increase of the major cell cycle switch pRb during melanoma development, 13 semiquantitative immunoreactivity of pRb significantly outweighed in nodular malignant melanomas compared to deep penetrating nevi. However, in contrast to these former observations also reporting a progressive increase of phosphorylated, inactive pRb, here, the differential expression of phosphopRb Ser795 lacked statistical significance, particularly due to broad intertumoral staining variations.…”

“…The determination of total expression scores (ES total , see Materials and methods) revealed a higher expression of pRb and phospho-pRb Ser795 in nodular malignant melanomas (mean ES totalÀpRb 143, mean ES totalÀpÀpRb Ser795 63) than in deep penetrating nevi (mean ES totalÀpRb 70, mean ES totalÀpÀpRb Ser795 53), a finding consistent with our latest data on common benign nevi vs nodular malignant melanomas 13 and with previous data on PCNA 12 (Figure 1a and b). Since this differential regulation was only significant for total pRb (P pRb o0.005, P pÀpRb Ser795 o0.1), applicability of the pRb/phospho-pRb ratio as discriminative marker for deep penetrating nevi remains doubtful.…”

“…However, in contrast to these former observations also reporting a progressive increase of phosphorylated, inactive pRb, here, the differential expression of phosphopRb Ser795 lacked statistical significance, particularly due to broad intertumoral staining variations. Strikingly, compared to the results on common benign nevi, 13 both pRb and phospho-pRb Ser795 showed a high deep dermal immunolocalization in a considerable subset of the deep penetrating nevus specimens indicating inactive pRb, that is a cell cycle permissive status, very similar to nodular malignant melanoma.…”

“…As reported recently by our group, a paradox progressive overexpression of the cell cycle controlling pRb and its increased inactivation due to phosphorylation are hallmarks of malignant melanomas. 13 According to our former observations, 13 there was an intratumoral heterogeneity of total pRb and phospho-pRb Ser795 expression in nodular malignant melanomas. Particularly at the subepidermal lateral and dermo-invasive portions, both markers revealed high expression scores in most samples (ES max 4100).…”

“…13,14 Briefly, the number of positive stained nuclei was estimated as percentage of all nuclei (p) per field of vision. In addition, the staining intensities (i) of positive cells were scaled from grade 1, when the cells showed a slight granular staining pattern, to grade 4, when the cells had completely filled nuclei.…”