Melanoma is one of the most prevalent malignancies and has a very poor prognosis. Mutations in v-raf murine sarcoma viral oncogene homolog B1 (
The high frequency of
Of note, in addition to melanomas,
Indirect evidence from cultured cells and animal models reveal that there may be a cooperative role between the constitutive activation of ERK pathway and the loss of p16 in tumor progression. Daniotti et al.  reported the co-existence of
There is evidence that treatment response to BRAFi and MEKi relies on a functional p16-cyclin D-CDK4-retinoblastoma (RB) axis.
As shown in Fig. 4, in addition to BRAF and MEK inhibitors, several drugs designed to inhibit the activity of CDK4 are in active clinical trials for melanoma and other cancers including LEE011 (Novartis Pharmaceuticals ), LY2835219 (Eli Lilly and Company), PD-0332991 (Pfizer) (http://clinicaltrials.gov/).
Deregulation of the p16-cyclin D:cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in malignancy including melanoma [12, 13, 39] and represents another attractive target in melanoma treatment. The great majority of melanoma cells have lost or reduced expression of wild-type
Various resistance mechanisms have been identified that contribute to treatment failure of melanoma patients to BRAFi and MEKi therapy. Loss of p16 may represent a common gateway permitting the phenotypic expression of several resistance mechanisms to BRAFi and MEKi (Figs. 1 and 3), a hypothesis that has not been and is waiting to be tested in clinical trials. We reported that simultaneous expression of
MEK inhibitor PD98059 (Calbiochem, San Diego, CA) was dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution, aliquoted and stored at -20C. CDK4 inhibitor 219476 (Cat. # 219476, Calbiochem, San Diego, CA) was dissolved in DMSO as a 2 mM stock solution and stored at 4C. Human melanoma cell lines 624Mel, A101D, and OM431 were kindly provided by Dr. Stuart Aaronson (Mount Sinai School of Medicine, New York, NY). Cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and 50 units/mL penicillin–streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator at 37C with 5% CO2. CellTiter 96® R AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega Corporation, Madison, WI) was used to measure dehydrogenase enzyme activity found in metabolically active cells. Melanoma cells were seeded in a 96 well plate at a density of 2 ×104 cells/well in DMEM with 5% FBS. On the second day, the culture medium in each well was changed to 150
Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) method using
For Western blotting, 1 × 106 melanoma cells were seeded in a cell culture dish (10 cm) in DMEM containing 5% FBS and antibiotics. On the second day, cells were treated with PD98059 and 219476 at the same concentration as described in the MTS assay. For cell cycle regulators cyclin-dependent kinase inhibitor p27 kinase interacting protein 1 (KIP1) and RB, cells were treated with the chemicals in medium with 5% FBS for 24 hr and then harvested. For apoptosis-related protein B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2), BCL2-like 1 (BCL2L1 or bcl-xL), inhibitor of apoptosis family (IAP) protein baculoviral IAP repeat-containing 5 (BIRC5 or survivin), apoptosis facilitator BCL2 interacting mediator (BIM), cysteine-aspartic acid protease (caspase) 3, and poly (ADP-ribose) polymerase (PARP), cells were treated with the various chemicals in DMEM with 5% FBS for 48 hr and then harvested. For phospho- and total-ERK, cells were treated with the chemicals in medium with 0.5% FBS for 18 hr and then harvested. Western blots were performed as described [1-3]. Briefly, harvested cells were lysed in Lysis Solution (Cell Signaling, Danvers, MA) supplemented with Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics Corporation, Indianapolis, IN). Protein concentration of lysates was determined using the Quick Start Bradford 1 × Dye Reagent (Bio-Rad, Hercules, CA). Lysates were separated in either 12 or 15% SDS-polyacrylamide gel, electrophoretically transferred to Immobilon-P membrane (Millipore Corp, Billerica, MA), and probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: BCL2 and tubulin, beta (Sigma-Aldrich, St. Louis, MO); BCL2L1 and BIRC5 (Santa Cruz Biotechnology, Santa Cruz, CA); phosphor-ERK, total ERK, Caspase 3, PARP, and PhosphoPlus(R) RB (Ser780, Ser795, Ser807/811) Antibody Kit (Cell Signaling, Boston, MA); p27KIP1 (BD Biosciences, San Jose, CA); and peroxidase-conjugated antimouse and antirabbit secondary antibodies (Calbiochem, San Diego, CA). Immunoreactive bands were visualized with SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The blots were exposed to blue sensitive blue X-ray film (Phenix Research, Candler, NC) [1-3].
We have shown previously that human melanoma cell lines 624Mel, A101D, and OM431 cell lines harbor heterozygous
PD98059 and 219476 inhibit tumor cell growth in a dose dependent manner [1, 2]. In order to make it possible to monitor the additional therapeutic effects of the combinatorial treatment, both chemicals were used at dosages lower than that which would lead to maximal effect by either agent. The cytotoxicity of PD98059 and 219476 was examined 24 and 48 hr after treatment using the MTS assay that measures the dehydrogenase enzyme activity found in metabolically active cells. After 24-hr treatment, there was no significant difference in cell viability between control, single, and combined treatment groups of 624Mel cells (
Next, we performed the TUNEL DNA fragmentation assay to identify loss of viability due to programmed cell death after 48-hr treatment. As shown in Figure 3, at the drug concentrations used, significantly different levels of apoptosis exist among control for PD98059, 219476, and combinatorial treatment groups (
As apoptosis was the major effect observed when melanoma cells were exposed simultaneously to MEK and CDK4 inhibitors, we examined the expression of several pro-apoptotic and anti-apoptotic proteins. Mono-treatment with PD98059 or 219476 caused a decreased or no change in the expression of anti-apoptotic proteins BCL2, BCL2L1, and BIRC5. While there were variations in the patterns of expression of BCL2, BCL2L1, and BIRC5 among the different cell lines (Fig. 8), combinatorial treatment caused a comprehensive down-regulation of the proteins in all three cell lines (Fig. 8). In addition, apoptosis facilitator BIM-EL was increased following treatment with PD98059 and PD98059 plus 219476 in all three cell lines. It was also increased in OM431 cells following treatment with 219476. Consistent with increased apoptosis, caspase 3 was activated by simultaneous treatment with PD98059 plus 219476 in all three cell lines, as shown by decreased procaspase 3, increased levels of the active form of caspase 3 (cleaved caspase 3), and degradation of PARP, a direct substrate of active caspase 3 (Fig. 8).
In this study, we simultaneously inhibited MEK and CDK4 kinases using pharmacological inhibitors PD98059 and 219476 and observed significantly increased apoptosis compared to control and single agent treatment. This is consistent with our previous report that simultaneous knockdown of BRAF using small interfering RNA (siRNA) and expression of
Apoptosis resistance is a critical factor for therapy failure in melanoma patients. Encouragingly, combined treatment with PD98059 and 219476 leads to significant apoptosis in all the three melanoma cell lines studied (Fig. 7). The apoptotic rate caused by the combined treatment is higher than the combined apoptosis by monotreatment, suggesting that MEK and CDK4 kinases mediate each other’s pro-survival effect. The apoptotic effect is associated with changes of apoptosis-related proteins (Fig. 8). PD98059 and 219476 combined treatment leads to significant down-regulation of the pro-survival proteins BCL2, BCL2L1, and BIRC5, and up-regulation of the pro-apoptotic protein BIM. We showed previously that BCL2 and BIM were regulated by
Genetic and epigenetic changes of
|Various mutations||Heterogeneous sequence changes|
|Bi-allelic deletion||Protein null|
|Promoter hypermethylation||Lower levels of p16|
As examples, Table 2 is a list of molecular assays to comprehensively examine
||Sanger sequencing||[86, 87]|
||Pyrosequencing (PyroMark Q24 CpG p16 Kit, Qiagen, Valencia, CA)||[82, 88, 89]|
|p16 expression||Immunohistochemical staining (IHC)||[90, 91]|
These assays need to be validated both technically and clinically with defined cut-off values. There should be correlation of results among assay methods; for example, cells with bi-allelic
|Lesion +ve||A||B||Positive predictive value = A / (A + B)|
|Lesion -ve||C||D||Negative predictive value = D / (C + D)|
|Sensitivity = A / (A + C)||Specificity = D / (B + D)|
Patients with metastatic melanoma have a median survival of 6-8 months . Recently, ipilimumab (Yervoy, Bristol-Myers Squibb), an inhibitor of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and vemurafenib (PLX4032, Zelboraf, Plexxikon/Roche), an inhibitor of mutant BRAF, gained FDA approval to treat patients with metastatic melanoma. Although both drugs offer new approaches to the treatment of advanced melanoma, their therapeutic efficacy is limited. Both drugs typically lengthen life by only several months in patients that initially responded to the treatment [94, 95]. There is mounting evidence that acquired resistance to BRAFi frequently correlates with reactivation of the RAS-RAF-MEK-ERK signaling pathway [52, 53, 64]. This finding led to clinical trials combining BRAFi and MEKi in patients with
ASK1: apoptosis signal-regulating kinase-1
ARF: alternative open reading frame
BCL2: B-cell chronic lymphocytic leukemia/lymphoma 2
BCL2L1: BCL2-like 1
BIM: BCL2 interacting mediator
BIRC5: baculoviral IAP repeat-containing 5, also known as survivin
BRAFi: BRAF inhibitor
Caspase: cysteine-aspartic acid protease
CDK2: cyclin-dependent kinase 2
CDK4: cyclin-dependent kinase 4
CDK4i: CDK4 inhibitor
CEP9: chromosome 9 centromeric probe
CLL: chronic lymphocytic leukemia
DMEM: Dulbecco's modified Eagle medium
DMSO: dimethyl sulfoxide
ERK: extracellular-signal-regulated kinase
FBS: fetal bovine serum
FDA: Food and Drug Administration
FGF: fibroblast growth factor
FITC: fluorescein isothiocyanate
HGF: hepatocyte growth factor
IAP: inhibitor of apoptosis family
IHC: immunohistochemical staining
KIP1: kinase interacting protein 1
LOH: loss of heterozygosity
MEK: mitogen-activated protein kinase/ERK kinase
MEKi: MEK inhibitor
MST2: sterile 20- like-kinase 2
PAGE: polyacrylamide gel electrophoresis
PARP: poly (ADP-ribose) polymerase
PBS: phosphate buffered saline
PI3K: phosphatidylinositol 3-kinase
RAF: v-raf murine sarcoma viral oncogene homolog. Human has three RAF: CRAF, BRAF, and ARAF
RAS: rat sarcoma viral oncogene homolog. Human has three RAS: HRAS, NRAS, and KRAS (KRAS4A and KRAS4B proteins arise from alternative splicing)
RB: retinoblastoma proteins including pRB, p107, and p103
SDS: sodium dodecyl sulfate
siRNA: small interfering RNA
TdT: terminal deoxynucleotidyl transferase
TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
UV: ultra violate
We thank Dr. Stuart Aaronson for human melanoma cell lines. This work was supported by Bill Walter III Melanoma Research Fund, Harry J. Lloyd Charitable Trust, and Cancer and Leukemia Group B Foundation (to J.D.).
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