Melanoma Cell Lines & Mutations


Tumor modelling facilitates the study of tumor growth in the tumor microenvironment. Therapies that affect the tumor microenvironment are critical for advancing the fight against cancer as emerging therapies target the network of signaling pathways essential for tumor growth and metastasis. Patient derived tumor models, including melanoma cell lines and tumor xenografts, are invaluable tools to study normal and malignant cells, tumor formation, and drug resistance. A diverse panel of melanoma cell lines is required to account for the varied genetic signatures of tumors, especially if the scientific goal is to establish drug sensitivity for tumor inhibition, therapy resistance, or tumor dormancy.

We produce, validate, and distribute a diverse collection of highly-characterized melanoma cell line models. These models are biologically stable in terms of gene expression pattern, tumor architecture, and mutation status, giving them the ability to predict clinical activity. These melanoma cell lines serve as individualized experimental models that are important for drug treatment and outcome. The collection, originally provided by Dr. Meenhard Herlyn of The Wistar Institute, has been characterized to confirm a match to that of the originating patient tumor tissue via mRNA, qPCR, and DNA short tandem repeat (STR) analysis. A summary table indicates the disease stage, gene mutations, pathology and relevant clinical data for each of these human melanoma cell lines. The key application of these cell lines include genetic studies, xenograft production, and drug testing and drug target discovery. In addition to viable cells, Rockland produces non-viable cells for lysate productiongenomic DNA, and total RNA from each melanoma cell line.  

 

Modeling Melanoma in Synthetic Skin

 

Figure 2.  Modeling melanoma in sythentic skin.  Image courtesy of Dr. Meenard Herlyn, Wistar Institute, Philadelphia, PA.  


>> Download: Melanoma Cell Lines Table with Microarray Gene Expression Analysis and STR Table

 


Patient Derived Cell Line

 

Figure 1.  Development of melanoma cell lines.  After a low number of cell passages, cells are banked and validated by extensive analysis including copy number determination, gene expression profiling, immunohistochemical staining with antibodies to known tumor biomarkers, STR and response to drugs.





Melanoma Mutations

Malignant melanoma is associated with genetic heterogeneity and a complex etiology.  In contrast to other skin cancers, melanoma has a strong tendency to metastasize with a consequently extremely poor overall prognosis for survival. The various phenotypes of melanoma are characterized by clinical features, such as bodily distribution or risk factors. Cutaneous, uveal, acral, and mucosal melanomas have divergent clinical courses and are associated with distinct mutations, and risk factors. In the majority of melanomas, a number of genes and signaling pathways are involved in cellular proliferation and growth, and have been implicated in the pathophysiology of melanoma.

We have organized our collection of melanoma cell lines based on the presence and/or absence of mutated or wild type genes. At a minimum we have characterized these cell lines for the following genes: BRAF, N-RAS, KIT, PTEN and CDK4.
 

Melanoma Microenvironment

 

Figure 3.  The melanoma microenvironment.  Image courtesy of Dr. Meenard Herlyn, Wistar Institute, Philadelphia, PA.  

                 
 Mutated BRAF Melanoma Cell Line

 

 Mutated N-RAS Melanoma Cell Line

 

 Mutated KIT Melanoma Cell Line

 

 Mutated PTEN Melanoma Cell Line

 

Mutated CDK4 Melanoma Cell Line

Melanoma Cell Lines with Mutated BRAF 

 

 

Melanoma Cell Lines with Mutated N-RAS 

 

 Melanoma Cell Lines with Mutated KIT 

 

 

Melanoma Cell Lines with Mutated PTEN 

 

 

Melanoma Cell Lines with Mutated CDK4 



 



BRAF (BRAFV600E) Mutations

Alterations in signaling cascades that are involved in cellular proliferation and growth have been implicated in the pathophysiology of melanoma. The mitogen-activated protein kinase (MAPK) pathway is a significant driver in melanoma and provides several possible targets for therapeutic intervention. In this pathway, the activation of RAS proteins stimulates the RAF kinases (ARAF, BRAF, and RAF1). The activation of RAF kinases leads to the phosphorylation of the MEK kinases, which in turn phosphorylate the ERK kinases. The RAS-RAF-MEK-ERK signaling pathway that impacts cellular proliferation, differentiation, and survival among many diverse cellular functions, is constitutively activated due to oncogenic mutations in the serine–threonine protein kinase, BRAF. Signaling is initiated when active RAS recruits RAF to the plasma membrane for activation through a complex process requiring lipid and protein binding, conformational changes, and regulatory phosphorylation and dephosphorylation events.




N-RAS Mutations 

NRAS-mutant melanoma is a distinct cohort of melanoma that comprises 15% to 20% of all melanomas and appears to confer a poor prognosis. NRAS mutations predominantly in codon 61, lead to increased cellular proliferation and are more potently tumorigenic. NRAS mutations are generally found among melanomas without BRAF mutations, and activating mutations in NRAS confer resistance to BRAF-targeted therapy. Mutations in N-RAS cause activation of downstream serine/threonine kinase MAPK through RAF. N-RAS mutations can also activate other pathways such as PI3K-AKT-MYC, which promote cell cycle progression, cellular transformation, and enhanced cell survival. Although direct inhibition of mutant N-RAS has not been effective, the targeting of downstream pathways such as MAPK pathway inhibitors may prove to be a useful treatment strategy. Furthermore, combined targeting of MEK and PI3K-AKT mammalian target of rapamycin (mTOR) pathways may effectively inhibit N-RAS-mutant melanoma, and may provide an alternative therapeutic approach.




KIT Mutations 

A small portion of melanomas have changes in a gene called C-KIT. KIT is a type III transmembrane receptor tyrosine kinase. Binding of its ligand, stem cell factor, results in receptor dimerization, autophosphorylation, and activation of several signaling pathways; thereby, mediating cancer cell growth, proliferation, invasion, metastasis, and inhibition of apoptosis. These gene changes are most common in mucosal melanomas derived from the genital regions or mutations in GNA11 or GNAQ genes in uveal melanomas. Some of the targetable mutations in the KIT gene are also found in acral and other mucosal (for example, penile or anal) melanomas but with lesser occurrence. Most KIT mutations are located in exon 11, which codes for the juxtamembrane domain, and in exon 13, which codes for a kinase domain.




PTEN Mutations 

PTEN (Phosphatase and tensin homolog deleted in from chromosome ten) is a tumor suppressor gene that is mutated in a large fraction of human melanomas. In melanoma, allelic loss or mutations of PTEN have been defined in 5–15% of uncultured melanoma specimens and metastases, as well as in 30–40% of established melanoma cell lines. An ectopic expression of PTEN in PTEN deficient melanoma cells has been shown to suppress growth, tumorigenicity and metastasis. The PTEN protein has both lipid phosphatase and protein phosphatase activity. The lipid phosphatase activity of PTEN decreases intracellular phosphatidylinositol (3, 4, 5) -trisphosphate level, ultimately preventing downstream AKT activity. PTEN, containing a phosphatase domain, is inactivated in 12% of melanomas through mutation or methylation.




CDK4 Mutations

The CDK4 is a tumor suppressor gene encoding a protein that helps control cell division. This gene is abnormal in about 5% of individuals with melanoma. CDK4 mutations are relatively common in melanomas in the extremities (such as limbs, fingers, and ears) and mucous membranes (such as those in the mouth, nose, and genitals). Affected individuals possess unique mutations; either an Arg24Cys, located in exon 2 of the gene or Arg24His mutation. The Arg24Cys mutated protein, which was first described as a tumor specific antigen in a human sporadic melanoma, specifically prevents binding of CDK4 protein to P16A. While there are no FDA-approved drugs that target CDK4, several CDK4 inhibitors are in the early stages of clinical trials.





Wistar Collaboration


Rockland has partnered with the Wistar Research Institute to produce, validate, and distribute a diverse panel of low passage melanoma cell lines from freshly excised metastases. More than 100 melanoma cell lines are grouped for BRAF, N-RAS, KIT, PTEN and CDK4 mutations. These preclinical tumor cell lines models can be used to identify the critical target genes and pathways enacted by genomic alterations and lead to more accurately prediction of the effectiveness of novel cancer therapeutics and facilitate cancer research.


 



The cellular and molecular heterogeneity of human cancers is well established. Different tumor cells can show distinct phenotypic and molecular characteristics of the original cancer, including chromosomal copy number variants, single nucleotide polymorphisms, and gene expression profiles. This heterogeneity has clinical implications in patient specific responses to therapy and represents a substantial challenge for cancer drug development. With the advent of precision medicine, i.e. explicit, targeted tumor therapies, it has become important to ascertain tumor subpopulations which respond to anti-cancer therapy. In this milieu, the tumor models that more accurately reflect patient tumor biology and provide the resolution necessary to represent the diversity of cancer patients, are required for testing novel anti-cancer compounds.



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