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From the Departments of Medical Biophysics*
and Medical
Oncology,
Sunnybrook Health Science Centre,
University of Toronto, Toronto, Ontario, Canada, and the Departments of
Tumor Biology
and
Pathology,§
Institute for Cancer Research, The
Norwegian Radium Hospital, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p21WAF1/CIP1 was the first mammalian cdk inhibitor to be identified and was found to be induced by wild-type p53 in response to DNA damage, thereby suggesting a role in the coordination of p53-mediated growth arrest and DNA repair.4,5 However, p21WAF1/CIP1 has also been demonstrated to be involved in cellular senescence, terminal differentiation, and apoptosis through p53-independent mechanisms.6-9 Notably, the gene encoding p21WAF1/CIP1 has been recently cloned and identified as a melanoma differentiating antigen (mda6), the expression of which is up-regulated in more differentiated melanoma cell lines and in melanocytes grown in vitro.10 In contrast, decreased p21WAF1/CIP1 mRNA and protein levels have been detected in cell lines derived from advanced melanomas and in tumor specimens obtained from melanoma metastases, as compared with the levels in early-stage malignant lesions, suggesting that loss of p21WAF1/CIP1 may contribute to the progression of this type of cancer.10,11
p27Kip1 was originally identified as a cdk inhibitor, the activity of which is up-regulated in vitro by transforming growth factor (TGF)-ß, by contact inhibition, or by serum depletion.12-15 Furthermore, p27Kip1 levels are increased during differentiation of cultured cells.16,17 p27Kip1 has been demonstrated to play an important role in regulating progression through G1 and entrance into the S phase of the cell cycle by binding to and preventing premature activation of cdk4/cyclin D and cdk2/cyclin E complexes.14,18 In addition, constitutive overexpression of p27Kip1 causes cell cycle arrest in the G1 phase. Recent studies have demonstrated that transgenic p27Kip1 knockout mice develop multi-organ hyperplasia and are larger than their normal littermates.19-21 This finding supports an important role for p27Kip1 in the negative regulation of normal cellular proliferation.
In contrast to the INK4 inhibitors, members of the KIP family, p21WAF1/CIP1 and p27Kip1, are rarely mutated in human cancer.22,23 The level of p27Kip1 has been shown to be regulated primarily at the post-transcriptional level through the ubiquitin-proteasome-mediated pathway.24 In colorectal carcinomas, decreased levels of p27Kip1 are associated with increased ubiquitin-mediated degradation of p27Kip1 and with a less favorable prognosis.25 Furthermore, low levels of p27Kip1 have also been associated with decreased survival of patients with breast,26-28 gastric,29 and non-small-cell lung cancer.30 These findings led us to study whether altered expression of p27Kip1 could also be detected in tumor samples obtained from patients with malignant melanoma and, if so, whether such alterations in p27Kip1 levels may have value as a prognostic marker. Recently, our panel of primary and metastatic melanomas has been used to analyze protein expression of p21WAF1/CIP1. In the aforementioned study, we observed reduced p21WAF1/CIP1 protein expression in advanced melanomas; however, no correlation between p21WAF1/CIP1 levels and clinical outcome was detected. In another recent study, by Porter et al,27 p27Kip1 and cyclin E proteins were both analyzed in a panel of breast carcinomas. The finding that low p27Kip1 and high cyclin E levels appeared to have a powerful synergistic effect as prognostic indicators spurred us to address whether analysis of p21WAF1/CIP1 and p27Kip1 levels together may be of greater prognostic value for melanoma than either alone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded tissue sections were obtained from 113 primary malignant melanomas, 45 melanoma metastases of which 36 had distant location, and 4 benign nevi. From 32 patients, both primary and metastatic material were collected. Of the primary tumors, 79 were classified as superficial and 34 as nodular. Clinical follow-up was available for 109 patients, and for 104 of these the combined value of p21WAF1/CIP1 and p27Kip1 could be examined.
Immunohistochemical Analysis
Sections of formalin-fixed, paraffin-embedded tissue were immunostained using the biotin-streptavidin-peroxidase method (Supersensitive Immunodetection System, LP000-UL, Biogenex, San Ramon, CA) and the Optimax Plus Automated cell staining System (Biogenex). Deparaffinized sections were microwaved in 10 mmol/L citrate buffer (pH 6.0) for 20 minutes (four times for 5 minutes each) to unmask epitopes and treated with 1% hydrogen peroxidase (H2O2) for 10 minutes to block endogenous peroxidase. The sections were subsequently incubated with monoclonal p27Kip1 antibody (K25020, Transduction Laboratories, Lexington, KY) diluted 1:200 (1.25 µg of IgG1/ml) for 30 minutes at room temperature and incubated thereafter with biotin-labeled secondary antibody (1:40) and streptavidin-peroxidase (1:40) for 20 minutes each. Tissue was stained for 5 minutes with 0.05% 3,3'-diaminobenzidine tetrahydrochloride freshly prepared in 0.05 mol/L Tris/HCl buffer, pH 7.6, containing 0.024% H2O2 and then counterstained with hematoxylin, dehydrated, and mounted in Diatex. All the dilutions of antibody, biotin-labeled secondary antibody, and streptavidin-peroxidase were made with phosphate-buffered saline, pH 7.4, containing 5% bovine serum albumin. All series included normal skin as positive controls. Negative controls included substitution of the monoclonal antibody with mouse myeloma protein of the same subclass and concentration as the monoclonal antibody. All controls gave satisfactory results. Four semiquantitative classes were used to describe the number of positive stained cells: -, none; +, <5%; ++, 5 to 50%; +++, >50%. The p27Kip1 staining was evaluated by one and, in cases that were not obvious, by two observers. In the latter cases a good concordance was always achieved. Based on previous studies, p27Kip1 protein expression was classified as high when more than 50% of the tumor cells stained positive25,26 or, alternatively, when more than 5% were p27Kip1 immunoreactive.
Statistical Analysis
The relationship between the expression of p27Kip1 and the median tumor thickness was evaluated nonparametrically using the Mann-Whitney two-sample test. Kaplan-Meier survival analysis was used to evaluate the relapse-free and overall survival data. A value of P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded tissue sections from 113 primary
and 45 metastatic malignant melanomas were immunohistochemically
analyzed for p27Kip1 protein expression. Nuclear
p27Kip1 staining was detected in 87% (98 of 113) of the
primary malignant melanomas (Figure 1A)
and in 67% (30 of 45) of the metastases (Figure 1B
; Table 1
). Furthermore, a higher fraction of
primary tumors as compared with metastatic lesions showed
p27Kip1 immunoreactivity in more than 5% of the cells
(54% versus 42%).
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. In another eight patients, with the same
proportion of cells stained in the primary and metastatic tumor, a
lower intensity of p27Kip1 staining was found in four of
the latter. Combining these results, altogether 18 of 29 (62%) of the
metastases expressed lower levels of p27Kip1 than the
corresponding primary tumor (Table 2)
. In
normal skin, distinct protein staining was present in all
nucleus-containing layers of the squamous epidermis and underlying
adnexal structures. A high p27Kip1 level was also observed
in the four benign nevi examined (Figure 1C)
.
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We found that there was no significant difference in the
distribution of low and high p27Kip1 staining between
nodular and superficial melanomas using either a 50% or a 5% cutoff
to describe high and low p27Kip1 levels (Table 1
and
results not shown). Interestingly, however, within the nodular subtype,
there was a significant variation in p27Kip1 staining as a
function of the vertical growth of the tumors, with lower levels in
thicker lesions (P = 0.05, 5% cutoff; Table 3
). Using a 50% cutoff to discriminate
between high and low p27Kip1 levels did not change the
results (P = 0.05, data not shown).
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).
Changing the cutoff (low p27Kip1 when <5% cells stained
positive) did not alter the outcome of the evaluation (data not shown).
When the superficial and the nodular spreading melanomas were examined
separately, there was a trend toward increased disease-free survival in
patients whose nodular tumor expressed higher levels of
p27Kip1 than those having less than 50% of the cells
stained positive for the inhibitor (P = 0.15;
Figure 2C
). Interestingly, when the cutoff between high and low
p27Kip1 protein expression was changed (high when >5% of
the cells were p27Kip1 positive), the disease-free survival
was clearly reduced for those patients expressing p27Kip1
in less than 5% of the tumor cells (P = 0.0002;
Figure 2D
). Notably, the level of p27Kip1 in the primary
tumor did not influence overall survival for patients with nodular
melanoma (P = 0.68 with 50% cutoff versus
P = 0.41 with 5% cutoff; data not shown).
p27Kip1 was not found to have any impact on either
disease-free or overall survival for patients with the superficial
spreading subtype (Figure 2E
and data not shown), and changing the
cutoff did not influence the outcome of the evaluation (Figure 2F
and
data not shown), possibly due to the long duration of follow-up
required for the demonstration of survival benefit for this subtype of
melanoma. Altogether, these results suggest that p27Kip1
may play different roles in the malignant progression of the two major
subgroups of melanoma, and furthermore, nodular melanomas, which
express virtually no p27Kip1 in the tumor cell nuclei, have
the highest risk of early recurrence.
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A total of 104 primary tumors and 31 metastases could be examined for expression of both p21WAF1/CIP1 and p27Kip1 proteins. Only one primary tumor showed positive immunoreactivity for both proteins in more than 50% of the cells. In contrast, 36 tumors (31%) showed both p27Kip1 and p21WAF1/CIP1 staining in fewer than 5% of the cells. High (>50% cells stained) staining of both proteins was never seen in metastatic samples, and indeed, 13 (42%) showed almost complete loss (<5% cells stained) of both p27Kip1 and p21WAF1/CIP1 staining.
When assessing the effect of reduced levels of both
p21WAF1/CIP1 and p27Kip1 proteins on clinical
outcome for the whole group of patients, we observed that moderate to
high levels of both proteins in the primary tumors did not correlate
with increased disease-free and overall survival (data not shown). When
pathological subgroups of melanoma were analyzed separately, however,
we found that, in nodular melanoma, low levels of
p21WAF1/CIP1 staining in cases that also showed reduced
p27Kip1 (<50% cells stained positive) was a stronger
predictor of relapse of disease (P = 0.01) than
low p27Kip1 alone (P = 0.15; Figures 3A and 2C
). However, with a 5% cutoff
between high and low p27Kip1 expression, less than 5%
p27Kip1-positive cells was still the strongest indicator of
early relapse (P = 0.0002), and
p21WAF1/CIP1 did not add any additional power to the
evaluation (P = 0.001; Figures 2D and 3C
).
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). In conclusion, we have shown that almost complete loss of p27Kip1 protein has prognostic value for relapse of disease for patients with nodular melanoma, but not for those with superficial malignant melanoma. p27Kip1 was not predictive of overall survival in either group. Furthermore, a combined analysis of p21WAF1/CIP1 and p27Kip1 proteins may have a greater prognostic potential for relapse-free survival in both of these pathological subgroups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with previous reports for normal tissue of epithelial origin,26,30,31 we found that p27Kip1 was highly expressed in normal skin, including keratinocytes, melanocytes, and benign nevi, supporting the notion that one important function of p27Kip1 may be to regulate quiescence in normal cells.32-34 This is in contrast to p21WAF1/CIP1, the expression of which is not detectable in melanocytes and benign nevi in vivo11,35 and which is also very low in G0-arrested melanoma cell lines in vitro.32 Thus, these two cdk inhibitors may play different roles in proliferation of cells of the melanocytic linage.
In agreement with what has been observed for other types of tumors,26,27,30,31 p27Kip1 was found to be heterogeneously expressed in malignant melanomas, with a trend toward less p27Kip1 immunoreactivity in metastatic lesions as compared with the levels seen in the corresponding primary tumors. However, when the panel of malignant melanomas was divided into the two most common pathological subtypes, the superficial and the nodular, important differences were observed. In nodular melanomas, thicker tumors had significantly less p27Kip1 than thinner, and potentially less aggressive, lesions. Furthermore, we found that patients whose tumors showed virtually no p27Kip1 immunoreactivity had a significantly shorter relapse-free period than those whose tumors expressed higher p27Kip1 levels. This finding is in accordance with recent reports indicating that p27Kip1 is a prognostic indicator of disease-free survival for patients with breast, colon, gastric, and non-small-cell lung cancers.25-30 Notably, Ki-67 immunostaining of a limited number of the melanomas revealed no clear correlation between the p27 levels and cell proliferation (data not shown), a finding that has been demonstrated also by others,25,26,36,37 suggesting that p27 may have additional functions that are not strictly related to cell cycle progression.
The most reliable prognostic marker for patients with melanoma is considered to be the vertical thickness of the primary tumor.38 Due to the low number of cases analyzed, we were, however, unable to determine whether the observed impact of p27Kip1 on disease-free survival for patients with nodular melanoma was independent of tumor thickness. Notably, p27Kip1 was not found to have any impact on overall survival for this group of patients. This could be due to the need for longer follow-up or to limitations in the power of the study. p27Kip1 has been suggested to have functions related to cell adhesion39-41 and may also play a role in tumor invasion.29 Based on these findings, reduced p27Kip1 levels may correlate with other events that favor metastasis formation by allowing cells to escape from the primary site.
In superficial melanomas, no association between p27Kip1 levels, tumor thickness, and relapse-free period could be detected. In contrast to nodular melanomas, the superficial spreading lesions are characterized by an initial radial growth phase that is generally considered to be significantly less aggressive than the vertical growth phase of superficial spreading melanoma.38 It may be speculated, therefore, that one reason for the observed differences in growth pattern between these two pathological subtypes may be related to differences in their responsiveness to altered levels of p27Kip1.
We next evaluated the prognostic potential of combined analysis of p21WAF1/CIP1 and p27Kip1. In nodular melanomas, low p21WAF1/CIP1 levels in combination with less than 50% of the cells showing p27Kip1 immunoreactivity was a better indicator of disease failure (P = 0.01) than p27Kip1 alone (P = 0.15). In this regard, it is of note that Porter et al27 found that a combined analysis of p27Kip1 and cyclin E had a greater prognostic value than either factor alone. However, the strongest predictor of disease-free survival for patients with nodular melanoma was the virtual complete loss of p27Kip1 protein (<5% tumor cells stained, P = 0.0002) as compared with those having higher levels of the cdk inhibitor. Altogether, our results suggest that p27Kip1 is a more important predictor of the aggressiveness of nodular melanomas than p21WAF1/CIP1.
We have shown previously that the protein level of p21WAF1/CIP1 increases with tumor thickness of superficial melanomas.11 One reason for the observed differences in aggressiveness between superficial spreading and nodular melanomas could be that p21WAF1/CIP1 functions to delay cell cycle progression and thereby tumor growth in the former. However, when evaluating the combined effect of p21WAF1/CIP1 and p27Kip1 in superficial melanomas, we found that moderate to high levels of p21WAF1/CIP1 (>5% cells stained) in combination with a low level of p27Kip1 (<5% cells stained) was a stronger indicator of disease recurrence than either inhibitor alone. Thus, in accordance with reports suggesting that p21WAF1/CIP1 may act as an assembling factor,42 it might be speculated that p21WAF1/CIP1 in early-stage superficial melanomas plays a role in activating cdk complexes, thereby facilitating cell cycle progression. Given the small sizes of the melanoma subsets studied, the prognostic potential of combined analysis of p21WAF1/CIP1 and p27Kip1 needs, however, to be verified in larger cohorts of both superficial or nodular melanoma subtypes.
In summary, our results suggest that virtually complete loss of p27Kip1 protein may have potential as an indicator of early relapse of disease in patients with nodular melanoma. Studies with larger sample sizes are needed to assess whether p27Kip1 has significant prognostic value independent of tumor thickness.
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Acknowledgements |
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Footnotes |
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Supported by stipends from The Norwegian Research Council (V. A. Flørenes) and Lillemor Grobstocks Legacy (V. A. Flørenes and G. M. Mælandsmo), by funding from the Norwegian Cancer Society (J. M. Nesland and R. Holm), and by grants from the National Institutes of Health (CA41223 to R. S. Kerbel) and the National Cancer Institute of Canada (to J. M. Slingerland). R. S. Kerbel is a Terry Fox Research Scientist of the National Cancer Institute of Canada. V. A. Flørenes was supported by a Postdoctoral Fellowship from the Medical Research Council of Canada.
Accepted for publication April 1, 1998.
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