The impact of the microenvironment on the expression of proteins that govern invasion and progression of melanoma is one of the fundamental, but yet largely unresolved questions regarding this malignancy. To gain a better understanding of the underlying molecular events involved in this process, the study presented here served to determine whether an in vivo skin organ culture system can recapitulate protein abundance differences between melanoma cells and surrounding skin of melanoma and adjacent non-involved skin tissue samples as identified by a differential proteomic analysis. The SOCs were injected with either VGP (WM983-A) or MGP (WM1158) melanoma cell lines, or collagen alone to serve as a control. These two melanoma cell lines are widely utilized and were selected solely to compare melanoma growth to normal (e.g. non-invovled) skin in this SOC model and not to provide a detailed comparison of VGP versus MGP. Approximately 10,000 cells were collected by laser microdissection from these FFPE SOC sections (). The microdissected cells were processed using an “MS-friendly” heat-induced, enzyme-mediated (HI/EM) antigen retrieval method, followed by trypsin digestion (). Peptide digests were analyzed by LC-MS/MS; shows a representative basepeak chromatogram of a digest from approximately 5,000 cells on column from the microdissected FFPE VGP melanoma SOC section. This analysis afforded the identification of upwards of several hundred proteins per sample at a false discovery rate of approximately 2%, with all samples processed in duplicate.
Mass spectrometry-based proteomic analysis of melanoma and skin organ cultures
Differences in protein abundance between the various SOC-derived samples were derived by spectral counting, where it was found that proteins such as tenascin-C (TN-C) and fibronectin, which have been described in the literature to play a role in melanoma cell invasion17, 18
, were identified by high spectral count in the MGP melanoma SOC sections, and at lower levels in the VGP melanoma SOC sections (). These proteins were either not identified or were identified at very low abundance in the the collagen and non-injected SOC controls. Increases in the abundance of TN-C have been documented in MGP melanomas, and it has been suggested that relatively low levels of TN-C expression may be associated with a lower risk for metastasis19
. Like TN-C, fibronectin is another protein implicated in melanoma invasion. Specifically, it has been reported that these two proteins stimulate the invasive features of primary melanoma cells in 3-D collagen matrices, and that TN-C, fibronectin and procollagen I form specific channel structures for melanoma invasion18
. Tenascin-C and fibronectin are produced not only by fibroblasts, but also by endothelial cells and keratinocytes20
. However, it is not yet fully established to what extent these proteins are also produced by VGP and/or MGP melanoma cells. The data presented here document that melanocytes, propagated in vitro
, do not produce detectable levels of TN-C () and only low levels of fibronectin (data not shown). In contrast, both MGP and VGP melanoma cells express substantial amounts of these matrix proteins. These matrix components are not only secreted but also incorporated into an insoluble matrix surrounding the cells, likely giving rise to the apparent difference in the relative levels of TN-C transcript versus protein abundances measured in this study. The important point regarding this finding is that these melanoma cells produce these matrix components which are known to drive cell migration and tumor invasion21
Spectral count data for selected extracellular matrix and cytoskeletal proteins identified in melanoma-injected or control SOCs and from melanoma and normal human skin tissue.
Expression of tenascin-C in melanoma cells
α-Actinin-4 (ACN4) is another protein identified as differentially abundant between the SOC melanoma samples and controls. Furthermore, as depicted in , this protein is clearly present at higher levels in melanoma cells compared to melanocytes, both at the level of the transcript and protein. α-Actinin-4 is an actin cytoskeleton filament bundling protein and plays crucial roles in cell migration and cytokinesis22
. In addition, growth factor signaling is important for dynamic control of ACN4/actin filament interactions, which in turn regulates cell adhesion during substratum attachment and nuclear segregation during mitosis23
Alpha-actinin-4 expression melanoma cells
The increased expression of both TN-C and ACN4 was verified by immunohistochemistry (IHC) in WM983-A injected SOCs in the region of the tumor lesion as compared to the collagen-injected controls (). As expected, TN-C appears to be localized to the extracellular space () whereas ACN4 expression is primarily observed to be intracellular (). While low levels of TN-C can be observed in the collagen-injected SOC, this observation likely arises from the fact that these skin cultures are immature and composed of neonatal fibroblasts and kerationcytes, both of which produce TN-C.
Immunohistochemical analysis of tenascin-C and α-actinin-4 in SOCs
Thrombospondin-1 (TSP-1) was identified with an elevated abundance in both the MGP and VGP SOC sections compared to the controls. TSP-1 is a secreted protein that plays a role in tissue remodeling, is upregulated in response to injury and inflammation24
and has been detected at elevated levels in sera of patients with advanced melanoma25–27
. Plectin was also substantially more abundant in the MGP melanoma cells than in the VGP melanoma cells. Plectin, a structural protein found in nearly every cell type, interacts with numerous cytoskelatal components and has a role in the interactions of intracellular junctions and contributes to tissue integrity28, 29
. Plectin has been shown to be upregulated in pancreatic and colon cancer30, 31
, but there are no reports that cite this protein as being expressed in advanced melanomas. Similar to tenascin-C and fibronectin, plectin was identified with an increased relative abundance in the melanoma tissue sample as compared to that of the normal skin sample.
Alpha-enolase and pyruvate kinase M2 were identified with elevated abundances in the MGP SOC section relative to the other SOC samples. Increased levels of α-enolase have been previously associated with tumor cell migration and metastasis32
and has been shown to be elevated in several metastatic melanoma cell lines33, 34
. This increased expression level of α-enolase and pyruvate kinase M2 may reflect the apparent increased utilization of glycolysis for ATP generation in tumor cells as suggested by Warburg35
There are a number of notable proteins identified in the present study that merit further investigation. Of these, transgelin 2, a homolog of transgelin and member of a family of actin-binding proteins that is proposed to be involved in cytoskeletal cross-linking and polymerization, was identified in greater relative abundance in the MGP SOC tissue section as compared to the other samples. There are several reports citing an upregulation of transgelins in gastric36
cancers. Transgelin has been shown to be involved with ERK-related signal transduction, however, the function of transgelin in cancer development and progression remains to be elucidated. The cytokeratin (CK) pairs 5/14 and 6/16 and 17 were observed with decreased abundances in the VGP SOC section. It is well known that cytokeratins play a significant role in the organization and integrity of cellular structure and are indicators for differentiation state and metastatic nature of tumor cells. A previous study established diminished expression levels of CK 5/14 and 6/16 and 17 in metastatic melanoma38
. The present analyses also reveal an apparent increase in the abundance of pulmonary surfactant protein D in the VGP SOC section. While its role in melanoma has not been fully explored, the gene encoding this protein has been shown to be induced in a variety of metastatic pulmonary and non-small cell lung cancers, and is detectable in a variety of others, such as gastric, pancreas and prostate39