Ching-Ni Njauw

Epha2 is a Critical Oncogene in Melanoma

EphA2 is a member of the Eph family of receptor tyrosine kinases and is highly expressed in many aggressive cancer types, including melanoma. We recently showed that EphA2 is also upregulated by ultraviolet radiation and is able to induce apoptosis. These findings suggest that EphA2 may have different, even paradoxical, effects on viability depending on the cellular context and that EphA2 mediates a delicate balance between life and death of the cell. To functionally clarify EphA2s role in melanoma, we analyzed a panel of melanoma cell lines and found that EphA2 levels are elevated in a significant fraction of the samples. Specific depletion of EphA2 in high-expressing melanoma cells using short hairpin RNA led to profound reductions in cellular viability, colony formation and migration in vitro and a dramatic loss of tumorigenic potential in vivo. Stable introduction of EphA2 into low-expressing cell lines enhanced proliferation, colony formation and migration, further supporting its pro-malignant phenotype. Interestingly, transient expression of EphA2 and/or BrafV600E in non-transformed melanocytes led to significant and additive apoptosis. These results verify that EphA2 is an important oncogene and potentially a common source of ‘addiction’ for many melanoma cells. Moreover, acute induction of EphA2 may purge genetically susceptible cells, thereby uncovering a more aggressive population that is in fact dependent on the oncogene.

EphA2 Is an Essential Mediator of UV Radiation–Induced Apoptosis

One of the physiologic consequences of excessive UV radiation (UVR) exposure is apoptosis. This critical response serves to eliminate genetically injured cells and arises, in part, from activation of DNA damage and p53 signaling. Other contributory pathways, however, likely exist but have not been fully characterized. In a recent global screen of UVR response genes in melanocytes, we identified the receptor tyrosine kinase EPHA2. Using a combination of genetic and pharmacologic approaches, we set out to investigate the upstream regulation of EphA2 by UVR and the functional consequences of this effect. We found that the UVR-associated increase in EphA2 occurs in melanocytes, keratinocytes, and fibroblasts from both human and murine sources. More specifically, UVR effectively up-regulated EphA2 individually in p53-null, p63-null, and p73-null murine embryonic fibroblasts (MEF), suggesting that the p53 family of transcription factors is not essential for the observed effect. However, inhibition of mitogen-activated protein kinase (MAPK) signaling by U0126 and PD98059 significantly reduced the UVR response whereas overexpression of oncogenic NRAS led to an increase in EphA2. These results confirm that UVR induces EphA2 by a p53-independent, but MAPK-dependent, mechanism. In response to UV irradiation, Epha2(-/-) MEFs were highly resistant to UVR-mediated cytotoxicity and apoptosis whereas introduction of EphA2 into both wild-type and p53-null MEFs led to activation of an apoptotic program that can be blocked by caspase-8 inhibition. These functional findings suggest that EphA2 is in fact an essential p53-independent, caspase-8-dependent proapoptotic factor induced by UVR.

Vemurafenib Synergizes with Nutlin-3 to Deplete Survivin and Suppresses Melanoma Viability and Tumor Growth

Purpose: For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition. Experimental Design: Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib. Results: Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3′s effects fully and vemurafenibs effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy. Conclusion: These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target. Clin Cancer Res; 19(16); 4383–91. ©2013 AACR.

MITF Modulates Therapeutic Resistance through EGFR Signaling.

Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF(V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs) though the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR induced vemurafenib resistance is ligand dependent. We then employed whole-genome expression analysis and discovererd that vemurafenib resistance correlated with the loss of MITF, along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients’ tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an “autocrine drug resistance loop” is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition.

Agminated Segmental Nevi Demonstrating Intranevic Concordance of BRAF Status

TO THE EDITOR Grouped patterns of pigmented lesions are infrequent. Here, we analyze a rare case of segmentally distributed, agminated nevi characterized by multiple densely clustered lesions that are confined to a developmental segment. In fact, most segmental nevi are not agminated (Happle, 2002) and arise because of perturbations in the proliferation, migration, and differentiation of embryological precursors, including melanocytes (Misago et al., 1991; Sun and Tsao, 2008). The unusual growth pattern suggests mosaicism, i.e., a condition whereby an organism is composed of two genetically distinct cell populations due to a post-zygotic mutation (Itin and Burger, 2009). Cutaneous mosaicism often manifests as lines of Blaschko, a checkerboard pattern, or a phylloid (leaf-like) pattern (Happle, 1993). Recently, mutational analyses of melanocytic nevi have revealed that congenital moles, common acquired nevi, Spitz nevi, and blue nevi are associated with significant mutation rates in NRAS (Bauer et al., 2007), BRAF (Pollock et al., 2003), HRAS (Da Forno et al., 2009), and GNAQ (Van Raamsdonk et al., 2009), respectively. These findings suggest that mutational activation of specific signaling molecules drives the formation of these nevi. We thus hypothesized that our patient’s agminated segmental nevi (ASN) resulted from a mosaic event related to one of the known RAS effectors. The patient is a 58-year-old man who developed numerous grouped nevi on his right leg shortly after birth. There was no other significant personal medical history or family history of melanoma or dysplastic nevus syndrome. On examination of his right thigh and calf, there was an extensive band-like cluster of >100 pigmented macules, plaques, and papules clinically consistent with junctional, compound, or intradermal nevi (Figure 1a). Background skin in the area showed no hypo- or hyperpigmentation. Over multiple visits, 11 clinically atypical nevi within the segment were removed although none had histological features of melanoma. Pathological evaluation of the biopsies largely showed intradermal nevi with congenital patterns (Figure 1b). Figure 1 Composite diagram of the clinical, histopathological, and molecular findings of the agminated segmental nevi (ASN). (a) Body chart illustrating the distribution of the ASN on the right postero-medial thigh and calf, it also illustrates the non-segmental ... This genetic study was approved by the Research Ethics Board at Dalhousie University, and was conducted according to Declaration of Helsinki Principles. The patient also gave his written informed consent. We analyzed the 11 ASN and 1 unrelated mole from the right upper arm, which was excised independently because of its clinical atypia. These 12 formalin-fixed, paraffin-embedded specimens were micro-dissected and screened for mutations in NRAS exons 2 and 3, and BRAF exons 11 and 15. There were no canonical activating mutations in NRAS exons or BRAF exon 11. However, all 11 ASN harbored the identical BRAFT1799A transversion (i.e., the BrafV600E alteration) in exon 15. (Primer set: 5′-AGATCTACTGTTTTCCTTTACTTACTACAC-3′ and 5′-GGCCAAAAATTTAATCAGTGGA-3′). This mutation was notably absent in the non-segmental nevus and in the germline DNA from the patient’s blood. During the course of Sanger sequencing, several samples had minor, but reproducible “A” peaks. To create a cross-validating platform, we devised a liquid bead-based assay to more precisely measure the intratumoral representation of the two BRAF alleles (i.e., wild-type “T” and mutated “A”). (Allele-specific primer for T1799T: 5′-CCACTCCATCGAGATTTCT-3′; and T1799A: 5′-CCACTCCATCGAGATTTCA-3′). The bead assay substantiated the presence of the mutation in several samples (Figure 1c). There are at least two molecular models that could explain our patient’s pigmented lesions. In a “driver” model, a mutation, such as BrafV600E, occurred as a mosaic event, and therefore triggers the transient proliferation of segmental melanocytes, i.e., mole formation; every ASN should then harbor the same BrafV600E alteration. Our finding of 100% mutational concordance among the 11 lesions is certainly consistent with this “driver” model. However, a second “mutator” model can also be considered if the mosaic event did not occur in the BRAF gene, but rather, at another locus which then predisposes the affected melanocyte to develop activating RAS pathway mutations, including some in BRAF. This model would have more support if the ASN were discordant for BRAF status or harbored distinct changes in BRAF, NRAS or some other gene. If the “mutator” model is in fact correct, 11 independent events would have been necessary to account for the observed concordance of BRAFT1799A mutations. How likely is this latter scenario? Bauer et al. recently found that 0/32 (0%) of true congenital nevi and 20/28 (71.4%) acquired melanocytic nevi “with a congenital pattern” harbored BRAFT1799A mutations. As the mosaic nature of the ASN is by definition present at birth, this man’s segmental nevus may be argued as a variation of a congenital nevus, and therefore, likely devoid of BRAF alterations. However, a more conservative approach would classify these moles as acquired nevi with a congenital pattern. In this case, the probability that independent BRAF mutagenesis occurred in 11/11 lesions examined would be estimated at (0.7111) or 2.3%, a low but certainly not negligible chance. There are two additional factors that must be considered in the calculus. First, the early onset and segmental nature of the ASN argues for a mechanistic link between the populating nevi. In other words, an analysis of 11 randomly distributed and arbitrarily selected adult-onset nevi from a single individual is quite different than an analysis from a single group of ASN. Second, there is a BRAF distinction between the nevi inside and the nevus outside of the segment. In this limited but important specimen, the formation of the non-segmental nevus is clearly triggered by a non-BRAF event. Though our evidence could be strengthened by a large-scale sampling of nevi from different sites, further molecular and statistical pursuits are not clinically tenable given the large number of surgeries that have already transpired. Recent reports suggest that BRAF mutations, at least in melanomas, occur more frequently on intermittently sunexposed skin and much less commonly in non-sun-exposed sites (i.e., mucosal and acral lentiginous melanomas) (Maldonado et al., 2003). The segmental nature of the ASN definitively eliminates solar participation in the activation of BRAF. Germline mutations in BRAF have also been reported in such conditions as cardio-facio-cutaneous (CFC) syndrome, a developmental disorder with characteristic facial, cardiac, and ectodermal defects (Niihori et al., 2006) However, most BRAF mutations in CFC are non-recurrent and rarely the oncogenic mutations observed in cancers (Rodriguez-Viciana et al., 2006). Perhaps the Braf signaling intensity associated with a germline V600E alteration is incompatible with life, whereas more tolerable mutations producing intermediate Braf activity exist in CFC. If the “driver” model is correct, the activating event may have passed a critical susceptibility period in utero. In summary, we describe a case of segmentally arranged, agminated nevi that exhibit BrafV600E concordance. Although the evidence strongly supports a “driver” mosaic model for the development of these lesions, the high prevalence of BRAF mutations in general raises the possibility of alternative models.

Abstract A157: Double‐targeted therapy against melanoma: p53 activation sensitizes melanoma cells to MEK inhibition

BRAF is mutated in approximately 70% of human melanomas. Although depletion of oncogenic BRAF resulted in tumor regression in mouse melanoma model, clinical development of anti‐BRAF signaling therapy has not been successful up to date. One of the possibilities is that human melanoma cells may use redundant signaling events to adapt to harsh environment induced by BRAF shutdown. p53 is a major tumor suppressor and is not mutated in most human melanomas, which makes p53 a valuble target for melanoma therapy development. Here, we show that p53 is inducible in most human melanoma cell lines in response to DNA damage. Nutlin‐3a, an MDM2 inhibitor, increases p53 protein expression as well as its activities in melanoma cells. Addition of nutlin‐3a significantly increases sensitivity of melanoma cells to MEK inhibitor U0126 induced cell death or cell cycle arrest. In addition, knockdown p53 by specific shRNAs effectively rescues apoptosis induced by MEK inhibitor and/or p53 activation. This data suggests that p53 activity is required for cell death induced by MEK inhibiors and/or p53 activators. In conclusion, p53 is a therapeutic target in human melanomas and its reactivation potentiates cell death induced by BRAF/MEK signaling inhibitory agents in p53 wild type melanomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A157.

Beta-catenin causes fibrotic changes in the extracellular matrix via upregulation of collagen I transcription

DEAR EDITOR, Keloid scars represent a pathological response to cutaneous injury. They are characterized by increased proliferation of fibroblasts, especially in the active growth phase, as well as an abnormally increased production of collagen. The Wnt/b-catenin signalling pathway is a multifunctional network that plays an essential role in embryonic development, organogenesis and tissue homeostasis. Aberrant Wnt signalling has been implicated in a variety of different diseases, including fibrotic disorders such as systemic sclerosis and keloid. The transforming growth factor (TGF)-b signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of the extracellular matrix in fibrotic diseases. TGF-b is also the major stimulus for the activation of the canonical Wnt pathway in fibrotic diseases. Wnt/b-catenin signalling is active in the presence of extracellular Wnt ligands, which induce a cascade of intracellular responses that stabilize cytoplasmic b-catenin. Stabilized b-catenin, the central transducer of the pathway, translocates to the nucleus and binds to Tcf/Lef family transcriptional cofactors to regulate target gene expression in a contextspecific manner. In this study, we hypothesized that expression of stabilized b-catenin in fibroblasts is sufficient to cause fibrotic changes in the extracellular matrix via upregulation of collagen I transcription. Firstly, we identified the expression of b-catenin and collagen in keloid tissues and cell lines. Then we generated a tetracycline-controlled stable immortalized fibroblast cell line expressing b-catenin to explore the role of stabilized b-catenin in collagen I transcription and synthesis. Immunohistochemical stainings for b-catenin, collagen I and collagen III were performed in 14 keloid and five normal tissues (Fig. 1a and Fig. S1; see Supporting Information). Of the 14 keloid tissues, nine (64%) and 13 (93%) showed moderate-to-strong staining for b-catenin and collagen I, respectively. In contrast, of the five normal tissues, four (80%) and five (100%) showed no/weak staining for b-catenin and collagen I, respectively. There was no significant difference in collagen III expression. Overall, fibroblasts in keloid tissues showed significantly higher expression of b-catenin (P = 0 046) and collagen I (P = 0 002) than those of normal tissues (Fig. 1b). The above results were confirmed by Western blot analyses showing increased b-catenin, collagen I and collagen III expression in keloid fibroblasts compared with normal dermal fibroblasts (Fig. 1c). To test the consequences of increased canonical Wnt signalling and b-catenin target validation in fibroblasts, an immortalized fibroblast cell line was engineered to express green fluorescent protein either alone or in conjunction with b-catenin under tetracycline control. Immortalized fibroblasts with b-catenin overexpression showed increased expression of b-catenin, collagen I and collagen III (Fig. 2a, b). Measurement of mRNA levels by reverse-transcriptase polymerase chain reaction showed that b-catenin (P = 0 02), collagen I (P = 0 007) and collagen III (P = 0 019) were markedly more expressed in immortalized fibroblasts with b-catenin overexpression compared with the immortalized fibroblast control (Fig. 2c). To determine whether b-catenin had a direct transcriptional effect on the collagen I promoter, we generated a COL1A2 luciferase reporter and observed a dose-dependent increase in luciferase activity with increasing amounts of b-catenin (Fig. 2d). Pathologically activated canonical Wnt signalling has been implicated in various fibrotic skin diseases. Given the prominent accumulation of b-catenin in keloid fibroblasts and keloid tissues and the crucial role of b-catenin in tissue fibrosis, we hypothesized that the stabilization of b-catenin in fibroblasts might have a direct effect on collagen I synthesis. We demonstrate in the present study that b-catenin and collagen I show increased expression in keloid fibroblasts. Staining for b-catenin and collagen I was observed significantly more in keloid tissues than in normal skin. Furthermore, overexpression of b-catenin resulted in increased transcription of COL1A2. The activation of the canonical Wnt pathway has a key role for fibroblast activation and collagen release in fibrosis, and collagen deposition in the dermis is a characteristic feature of keloid. Previous reports have shown that expression levels of extracellular matrix-encoding genes are responsive to expression of stabilized b-catenin. Consistently with our findings, Beyer et al. showed that fibroblast-specific stabilization of b-catenin resulted in rapid and progressive fibrosis with prominent dermal thickening, differentiation of resting fibroblasts into myofibroblasts and increased accumulation of collagens. In our study, we observed the role of b-catenin in the regulation of collagen I transcription. We found that the increased expression of b-catenin in fibroblasts is sufficient for increased collagen I synthesis, with concomitant increases in COL1A2

Aggressive skull base metastasis from uveal melanoma: a clinicopathologic study.

Purpose We present the clinical, pathologic, and genetic findings of the first reported case of choroidal melanoma that developed a late recurrence and aggressive metastasis to the skull base without evidence of hepatic involvement. Methods Retrospective chart review and clinicopathologic correlation of ocular and brain tissue, including sequencing of BAP1 for mutations. Results A 55-year-old woman was diagnosed with choroidal melanoma and treated with proton radiotherapy. Six years later, she developed a rapidly growing local recurrence involving the ciliary body and iris. Upon enucleation, histopathology revealed an iris and ciliary body epithelioid melanoma that was contiguous with the previously treated, regressed spindle cell choroidal melanoma. Imaging was initially negative for brain involvement. Two months later, she developed cranial neuropathies and was found to have a large skull base lesion that required surgical debulking for pain palliation. Histopathology confirmed the lesion to be metastatic melanoma. Both ocular and brain tumor specimens were wild-type for BAP1. Throughout her course, she developed no hepatic metastases. Conclusions Uveal melanoma may metastasize to the skull base. The present case was characterized by delayed onset and unusual aggressiveness of the metastatic disease, and lack of BAP1 mutation. The unusual course highlights a unique phenotype that may reflect an alternate molecular mechanism for metastatic disease.

CDKN2A/CDK4 Status in Greek Patients with Familial Melanoma and Association with Clinico-epidemiological Parameters

Approximately 5–10% of melanoma cases occur in a familial context. CDKN2A/CDK4 were the first high- penetrance melanoma genes identified. The aims of this study were to evaluate CDKN2A/CDK4 variants in Greek familial melanoma patients and to correlate the mutational status with specific clinico-epidemiological characteristics. A cross-sectional study was conducted by genotyping CDKN2A/CDK4 variants and selected MC1R polymorphisms in 52 melanoma-prone families. Descriptive statistics were calculated and comparisons were made using the X2 test, Fisher’s exact test and Student’s t-test for statistical analysis, as appropriate. CDKN2A variants were detected in 46.2% of melanoma-prone families, while a CDK4 variant was found in only one family. This study confirmed that, in the Greek population, the age at melanoma diagnosis was lower in patients carrying a variant in CDKN2A compared with wild-type patients. No statistically significant associations were found between CDKN2A mutational status and MC1R polymorphisms.

Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing

Background Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM). Methods Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three human melanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of human melanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided. Results Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10-6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10-4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10-4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10-5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels. Conclusions The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene.