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10.1245/s10434-006-9226-2
Annals of Surgical Oncology 14:1416-1423 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Down-Regulation of Pro-Apoptotic Genes is an Early Event in the Progression of Malignant Melanoma

Eric H. Jensen, MD1,2, James M. Lewis, MD1, James M. McLoughlin, MD1, Michael D. Alvarado, MD1, Adil Daud, MD1, Jane Messina, MD1, Steven Enkemann, PhD1, Timothy J. Yeatman, MD1, Vernon K. Sondak, MD1 and Adam I. Riker, MD1,3

1 Department of Interdisciplinary Oncology, Cutaneous Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Stabile Research Building, Room 22043, 12902 Magnolia Drive, Tampa, FL, 33612, USA
2 Department of Surgery, Division of Surgical Oncology, Mayo Mail Code 195, 420 Delaware Street, SE, Minneapolis, MN 55455, USA
3 University of South Alabama, Mitchell Cancer Institute, 307 North University Blvd, MSB 2015, Mobile, Alabama 36688 0002, USA

Correspondence: Address correspondence and reprint requests to: Adam I. Riker, MD; E-mail: ariker{at}southal.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Introduction: Down-regulation of apoptosis genes has been implicated in the development and progression of malignant melanoma. We used cDNA microarray to evaluate pro-apoptotic gene expression comparing normal skin to melanoma (thin and thick), nodal disease and distant metastases.

Methods: Twenty-eight specimens including skin (n = 1), thin melanoma (n = 6), thick melanoma (n = 7), nodal disease (n = 6), and distant metastases (n = 8), were harvested at the time of resection from 16 individuals. RNA was isolated and microarray analysis utilizing the Affymetrix GeneChip (54,000 genetic elements, U133A+B... levels) was performed. Mean level of expression was calculated for each gene within a sample group. Expression profiles were then compared between tissue groups. Student’s t-test was used to determine variance in expression between groups.

Results: We reviewed the expression of 54,000 genetic elements, of which 2,015 were found to have significantly altered expression. This represents 1,602 genes. Twenty-two pro-apoptotic genes were found to be down-regulated when compared to normal skin. Overall reduction was evaluated comparing normal skin to metastases with a range of 3.31–64.04-fold-decrease. When comparing the tissue types sequentially, the greatest fold-decrease in gene expression occurred when comparing skin to all melanomas (thin and thick) (p = 0.011). Subset analysis comparing normal skin to thin melanoma or thick melanoma, revealed the greatest component of overall reduction at the transition from thin to thick lesions (p = 0.003).

Conclusion: Sequential down-regulation of pro-apoptotic genes is associated with the progression of malignant melanoma. The greatest fold-decrease occurs in the transformation from thin to thick lesions.

Key Words: Apoptosis • Melanoma • Metastasis • Gene profiling • Microarray


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The incidence of newly diagnosed cases of melanoma continues to increase. Currently, melanoma is responsible for six out of every seven deaths from all diagnosed cutaneous malignancies, and is the fifth leading cause of cancer death in men and seventh in women.1 Although the majority of cases of primary cutaneous melanoma are localized and adequately treated with appropriate surgical resection, advanced disease continues to be exceedingly difficult to successfully treat. Furthermore, the survival for those who develop metastatic disease has not significantly changed in many years. Despite numerous efforts to enhance our therapeutic arsenal over the last decade, we continue to struggle with our understanding of the basic immunologic and molecular mechanisms involved in the progression of melanoma.

The advent of the human genome project has led to renewed interest in the evaluation of many cancers at a more fundamental molecular level. Gene microarray analysis and profiling has allowed for an unprecedented interrogation of almost all of the known human genome (~30,000 genes), providing a fresh insight into the genetic basis of cancer initiation and progression. Microarray technology has recently been widely used to create "gene fingerprints" for many cancer types, allowing for a clearer understanding of the genetic complexities involved, in addition to providing more accurate diagnosis, staging and prognostic variables.2–6 Similarly, many studies have shown promise in utilizing microarray analysis and profiling to evaluate the sensitivity of tumors to chemotherapy or other targeted approaches to therapy.7–10

It is our hope that the ability to characterize expressed genetic changes during melanoma progression may allow us to develop an improved understanding of this disease process at the molecular and genetic level. This may translate into improvements in the selection criteria used in the clinico-pathologic criteria and staging of individuals with melanoma. We hypothesize that such advancements will lead to improvements in both staging and treatment decision-making, all based upon the genetic signature of the individual patient with melanoma.

Cellular apoptosis refers to programmed cell death, which is inherent to all healthy tissues undergoing normal cellular turnover and growth. A feature that is common to most solid tumors is the loss of function of apoptotic mechanisms, with the dysregulation of cell growth as a defining characteristic of malignancy. In light of this, the goal of many therapeutic strategies (including chemotherapy and some biologic therapies) is the initiation of apoptosis, attempting to establish programmed cell death in tumor cells as a therapeutic intervention.11–15 Our aim in this study was therefore to evaluate the expression, and specifically the down-regulation, of pro-apoptotic genes during the hypothesized progression of primary melanoma to metastatic disease. Primary melanomas included all levels of Breslow’s thickness and meta-static disease encompassed bulky, tumor replaced lymph nodes as well as distant metastases.

One of the shortcomings of gene expression pro-filing has been the highly variable nature of gene expression between individuals with pathologically similar tumors. Caution should therefore be utilized when attempting to directly compare gene expression profiles between various individuals with similar cancers, recognizing that each individual with cancer is indeed unique with a unique cancer signature. However, certain insights can be gained by grouping similar individuals with melanoma, for instance, into either primary or metastatic disease, focusing on more global comparisons of gene expression. Such global comparisons may prove to be the most useful when examining the up and down-regulation of unique genes involved in the metastatic process. Lastly, even if we are able to evaluate the same lesions (primary and metastatic) from the same patient, we must still realize that there is a significant amount of tumor cell heterogeneity within individual tumor cell deposits, whether primary or metastatic, thus adding to the complexity of this problem. We have attempted to limit these confounding factors by pooling data from multiple patients and reporting the mean change in expression for each group.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
All investigations were performed with the approval of the University of South Florida Institutional Review Board for this protocol (MCC #13448, IRB #101751) and in accordance with all regulations regarding handling of patient tissues and information. Informed consent was signed by each patient who participated in this study.

Tumor samples were collected at the time of surgical resection of primary and metastatic melanoma samples. Once removed, samples were transported to the pathology department for evaluation by the pathologist and subsequent procurement and cryo-preservation of all samples within 5–10 min. For this study, a total of 28 representative specimens from 16 patients were utilized: normal skin (n = 1), thin (≤ 1.2 mm) melanoma (n = 6), thick (≥ 1.2 mm) melanoma (n = 7), macroscopic nodal disease (n = 6), and distant metastases (n = 8). Microdissection was not performed on these specimens prior to gene profiling as they were taken from gross tumor mass with no extraneous tissue at the time of harvesting. Total RNA was isolated from snap frozen specimens using Trizol reagent and standard isolation protocols (Invitrogen, Carlsbad, CA, USA). Secondary purification was performed using RNAEasy columns and quality of RNA was evaluated for evidence of degradation using a bio-analyzer prior to gene microarray analysis (Agilent Technologies, Palo Alto, CA, USA). Gene expression profiling was performed using the Affymetrix GeneChip (54,000 genetic elements, U133A+B).

Gene expression data was collected for each of the 28 samples. Mean expression was calculated for all genes within each tissue group (pro-apoptotic genes shown in Table 1Go). Fold change in expression was then calculated by dividing the expression of normal skin by the mean expression of the comparison group. Fold change values >1 therefore represent decrease in mean expression while fold changes <1 indicate increase in mean expression. A value equal to 1 indicates no change in gene expression between tissue types. Because normal biologic processes generally occur within twofold of average gene expression, we consider expression changes greater than twofold to be significant alterations.


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  		TABLE 1. Mean expression of pro-apoptotic genes is shown
 
Fold-changes in gene expression between tissue types (normal skin to thin melanoma, thin melanoma to thick melanoma, etc.) were then compared using Student’s t-test. Statistically significant differences in pro-apoptotic gene expression between tissue groups are identified by p-value <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Gene expression profiling identified 1,602 genes with significantly altered expression when comparing normal skin to primary melanoma (thin and thick), nodal disease and metastases. After review of gene ontogeny archives, we identified pro-apoptotic genes which were significantly (> 2-fold) down-regulated. This resulted in a total of 22 genes. Ten pro-apoptotic genes with the greatest down-regulation in gene expression were analyzed more closely (Table 1Go). We limited our evaluation to those genes, which have been described in the current literature as related to their primary role in the apoptotic process. Table 1Go shows the sequential down-regulation of pro-apoptotic genes when comparing normal skin to primary melanoma (thin and thick), nodal and distant metastases. The greatest difference in gene expression was found when comparing skin to that of primary melanoma. A similar, but less significant decrease in gene expression was uniformly identified when comparing primary melanoma to nodal disease. All but one gene (TNFRSF10) had the lowest overall gene expression when evaluating nodal disease. This was followed by a trend towards an increase in gene expression when comparing nodal disease to that of distant metastases. Figure 1Go illustrates overall pro-apoptotic gene expression comparing each of the melanoma tissue types. There is a dramatic reduction of gene expression when comparing thin to thick melanomas with very little further change noted when comparing nodal and metastatic tissues.


Figure 1
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  		FIG. 1. Sequential down-regulation of pro-apoptotic genes is shown. The greatest reduction in gene expression was identified when comparing thin to thick primary melanoma. No significant change occurred in nodal or distant metastases.

 
Table 2Go shows mean fold-decreases in pro-apoptotic gene expression when comparing the various sites of disease. Genes are listed in order of overall fold-decrease when comparing skin to metastatic disease. The greatest reduction in gene expression for all of our study genes was found when comparing skin to all melanomas [p = 0.011 (range 8–63.77-fold-decrease)]. Interestingly, subset analysis revealed the greatest proportion of this decrease to be between thin and thick melanomas (p = 0.003). Very little further decrease in expression was seen when comparing thick melanoma to nodal disease. As mentioned previously, a non-significant trend towards an increase in apoptotic gene expression was seen in the metastatic tissue group.


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  		TABLE 2. Fold-decrease in gene expression is shown, comparing each successive tissue type (i.e. skin to thin melanoma, thin to thick melanoma, thick melanoma to nodal metastasis, and nodal metastasis to distant metastasis)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
In this study, we have identified a group of genes involved in the cellular apoptosis pathway that are down-regulated during the hypothesized progression of a primary to metastatic melanoma. Multiple melanoma samples of each tissue type were evaluated in a parallel fashion with the pooled gene profiles directly compared in an e3ort to reduce the possibility of erroneous findings as a result of melanoma cell heterogeneity between patients. Although we originally hypothesized that pro-apoptotic genes may be down-regulated, the timing of such events in the process of melanoma progression were unknown. We have demonstrated that the down-regulation of pro-apoptotic gene expression occurs early in the development of invasive disease and coincides with progression from AJCC stages I to II primary melanoma. In our test group, we considered thin melanomas to include lesions ≤1.2 mm in Breslow’s depth. This was in recognition of a mislabeled specimen that was originally thought to be <1 mm in depth, but was in fact slightly deeper.

We have found that down-regulation of pro-apoptotic gene expression occurs very early in primary melanoma progression. These changes seem to coincide with the transition of stages I to II tumors, and are remarkably consistent between all genes evaluated. It can be inferred from these results that apoptosis mechanisms, while perhaps not central to the initiation of melanoma, may play an important role in the transition from a non-invasive primary cutaneous melanoma (radial growth phase) to one that has aquired the genes necessary for invasion and metastatic potential (vertical growth phase and beyond). The comparison between normal skin and thin melanoma revealed only a modest reduction in expression of apoptotic genes, while a more pronounced change was noted between thin and thick lesions. This finding lends support to the biologic basis for current AJCC staging guidelines distinguishing stages I and II primary tumors. It appears that the progression from thick primary to metastatic nodal and distant disease is accompanied by a less prominent reduction in pro-apoptotic gene expression than the transformation from thin to thick lesions. Interestingly, most of these genes showed a modest increase in gene expression when comparing nodal disease to distant metastases. We are examining the significance of this finding at present.

The greatest reduction in gene expression was found in the forkhead box gene family, specifically, the gene FOXQ1, which had a 64.04-fold-decrease when comparing skin to metastatic disease. The forkhead box gene family is a diverse group of transcription factors involved in development, metabolism, immunomodulation and cancer.16 Unlike most of the other genes evaluated in this study, the FOXQ1 had a gradual decrease in expression through the progression of tumor initiation, growth and metastases. In fact, the transition between skin to thin melanoma and thin to thick melanoma revealed very similar expression changes of –7.57-fold and –7.75-fold, respectively (Table 2Go). Similar to the other genes evaluated, further reduction was only minimal between nodal and distant disease. This may be indicative of a more prominent role in the initiation of invasive disease, although this will require further verification.

Tumor protein p73-like (TPL73L) and p53 regulated apoptosis-inducing protein 1 (P53AIP1) belong to a well-known family of tumor suppressor genes. Although p73 was only identified in 1997, it is likely ancestral to p53, a gene which has been extensively studied in its relationship to cancer development.17 Both of these genes seem to play a vital role in cellular apoptosis and are significantly down-regulated during the early progression of melanoma. TP73L had a 63.77-fold-decrease in expression comparing skin to melanoma, with a particular reduction between thin and thick lesions (Fig. 2Go). P53AIP1 had a 26.45-fold-decrease between thin and thick primaries. The p73 family of genes and its role in carcinogenesis has been studied in a wide variety of cancers, most of which have been found to have significant increases in gene expression.18–21 Only a few studies have identified down-regulation of p73, which seems to be related to hypermethylation silencing of transcription.22,23 In fact, p73 can induce apoptosis, even in the absence of functional p53, which has long been considered one of the central pro-apoptotic genes.24 Interestingly, p53 induced apoptosis in fibroblasts cannot proceed without functional p73.25 These findings indicate a prominent function of p73 in the control of programmed cell death. Here, we have observed a significant reduction in the expression of TP73L, the result of which is still yet to be determined.


Figure 2
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  		FIG. 2. Line graph representing overall gene expression of each tissue type. Note the uniformity of each curve, with substantial reduction noted when comparing thin to thick primary melanomas. Further alteration in expression comparing primary to nodal and distant metastases does not occur.

 
Tumor necrosis factor superfamily 10 (TNFSF10, TRAIL, APO-2 ligand) and tumor necrosis factor receptor superfamily 10 [TNFRSF10A, TRAIL-R1, Apo-2, death receptor 4 (DR4)] belong to a group of tumor necrosis related apoptosis inducing ligands and receptors (TRAIL, TRAIL-R1) which have been widely shown to play a role in cell death. Similarly, tumor necrosis factor receptor superfamily 25 (TNFRSF25, Apo-3, DR3, LARD) is recognized in its pro-apoptotic capacity. All of these related genes have been found to be significantly down-regulated in the current study (Fig. 2Go). The TNF receptor and ligand families have broad-reaching implications in the control of cell turnover, making the full scope of their function difficult to ascertain. Their role in the progression of melanoma has not been previously described.

TNFRSF25 has been shown to stimulate apoptosis via an NF-kappa B mechanism in HEK293 cells.26 The TNF-receptor family is a group of cysteine-rich cell surface receptors, which share a preserved intracellular domain, commonly referred to as the "death domain" because of its function in the control of cell death. Overall, the expression of TNFRSF25 was markedly reduced (21.74-fold-decrease) when comparing skin to metastatic melanoma (Table 2Go). Significantly, the majority of this alteration was seen comparing skin to melanoma (17.21-fold-decrease). Once again, the specific role of TNFRSF25 in melanoma has not been studied, but it is clearly an important regulator of normal cell turnover, which is altered during the progression of disease.

TNFS10 and TNFRSF10A are ligand–receptor members of the TRAIL family of genes. Multiple studies have investigated the role of TRAIL in melanoma apoptosis and few have also evaluated this pathway as a potential therapeutic target.29–34 Ras oncogenes, which are known to be involved in the melanogenesis pathway, have been shown to cause alterations in cellular proliferation through inhibition of apoptotic signaling. In colon cells, transformed Ras leads to uncontrolled cell proliferation, however, in the presence of TRAIL, apoptosis is induced via death receptor 4 (DR4, TNFRSF10A).35 Similarly, silencing of TNFRSF10A in ovarian cancer leads to TRAIL resistance and therefore diminished control of cellular proliferation.36 Alterations in the function of DR4 may also be related to increased risk of breast cancer.37 As our understanding of the TNF-related ligand and receptor families continues to expand, more focus will likely be placed on identifying potential therapeutic targets which may act to reestablish apoptotic mechanisms in a variety of cancers, including melanoma.

The pleomorphic adenoma gene-like 1 gene (PLAGL1, ZAC) is a zinc finger transcription factor that has been described to play a role as a tumor suppressor gene. Loss of function or down-regulation of expression has been identified in both breast and ovarian cancer cells lines.38,39 Gene profiling has similarly indicated down-regulation of PLAGL1 in multiple tumor types, including breast and lung carcinoma.40,41 The demethylating agent, azacytidine, has been shown to re-induce PLAGL1 expression (and apoptosis) in breast cancer cell lines, suggesting that promoter region hypermethylation is a likely culprit in the down-regulation of gene expression.38 Well-differentiated squamous cell skin cancers seem to retain expression of PLAGL1 while less differentiated basal cell tumors have a dramatic decrease in expression.42 In our present study, we have identified similar findings of significant diminution of PLAGL1 gene expression. Again, the largest fold-decrease (9.98-fold) occurs when comparing thin to thick melanoma (Fig. 2Go).

The caspase and calpain family of proteases have been shown to play a central role in a calcium dependant apoptotic pathway although the exact function of all of their members is not fully understood. We have identified caspase 14 (CASP14) and calpain small subunit 2 (CAPNS2) as significantly down-regulated during the progression of melanoma. Caspase 14 has typically been associated with a proteolytic/inflammatory role, which has not been well reported.43–45 The calpains are a closely related family of messenger proteins, believed to play a key role in the initiation and regulation of cell death. The calpain/caspase cascades share many commonalities although much is yet to be learned regarding the clinical relevance of their function.46

The PAWR (Par4) gene has been mapped to chromosome 12q21 and was first identified as part of the apoptotic pathway in prostate cells undergoing cell death after androgen withdrawal.47 It has also been shown to play a pro-apoptotic role in a melanoma cell line.48 Inactivation of PAWR has been shown to result in hyperactivation of NF-kB and impairment of JNK and p38 in mouse embryonic fibrablasts.49 The mechanism of this inactivation in vivo is likely related to DNA hypermethylation. There has been extensive research into the Ras/Raf/ MEK/ERK MAPK pathway and the development of melanoma. A Ras-dependant PAWR activity reduction has been identified in epithelial cells and seems to be the result of promoter methylation.50 It is clear from our observations that a significant decrease in PAWR expression occurs during melanoma progression, with an 8.37-fold-decrease at the thin-thick transition.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In this study, we have identified a group of pro-apoptotic genes sharing a uniform trend of sequential down-regulation of the genetic expression during the progression of malignant melanoma. In fact, when viewed simultaneously, the graphs representing pro-apoptotic gene expression for grouped stages of malignant melanoma look remarkably similar (Fig. 2Go). Our findings support a central role for the loss of apoptotic mechanisms in melanoma development. While modest down-regulation was seen in comparing skin to thin melanoma, the greatest proportion of down-regulation consistently occurred between thin and thick primaries.

As the invasiveness of a primary cutaneous melanoma proceeds, control of cellular proliferation is lost, as indicated by the drastic decrease in apoptotic gene expression. In contrast, nodal and distant metastases show very little additional reduction through the metastatic process. In fact, most of the genes evaluated revealed modest increases in expression as metastatic disease progressed. Further studies and follow-up will be required to determine the prognostic implications of these findings.

Our findings support the idea that biologic processes underlie current AJCC stage criteria. We have shown that significant genetic alterations are occurring at the same time (and may be at least partially responsible for) the progression from stages I to II disease. Although very little is known about the function of these pro-apoptotic genes in melanoma, most have been described in other cancers, lending support to a potentially important but undefined role.

Evaluation of pro-apoptotic genes as potential therapeutic targets in the treatment of melanoma will also require verification. We believe that with greater understanding of the molecular basis of disease progression, a unique insight will be gained into the primary events involved with the genetic progression of primary melanoma to metastatic melanoma. With such novel insight, it may then be possible to identify criteria for improving overall patient staging, treatment modalities and most importantly, patient outcome and improved survival.

Received for publication July 6, 2006. Accepted for publication July 7, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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