Cooperative interactions of PTEN deficiency and RAS activation in melanoma metastasis

Abstract

Melanoma displays frequent activation of RAS/RAF/MAPK and PI3K/AKT signaling pathways as well as inactivation of CDKN2A (INK4a/ARF) and PTEN tumor suppressors via genetic and epigenetic alterations. Pathogenetic roles of these melanoma-prone mutations and their genetic interactions have been established in genetically engineered mouse models. Here, we catalog frequent genetic alterations observed in human melanomas and describe mouse models of melanoma initiation and progression, including our recent study that investigated the genetic interactions of RAS activation and PTEN loss in a CDKN2A (INK4a/ARF) null melanoma prone genetic background. We showed that loss of PTEN cooperates with HRAS activation, leading to increased development of melanoma and emergence of metastasis. Moreover, we observed that RNA i-mediated PTEN inactivation in RAS-driven melanomas enhanced migration and invasion with concomitant downregulation of E-cadherin, the major regulator of epithelial and mesenchymal transition, and enhanced AKT2 phosphorylation, which has been previously linked to invasion and metastasis of several cancer types, including breast and ovary. These data show that activated RAS cooperates with PTEN loss in melanoma genesis and progression.

Melanoma is the most aggressive form of skin cancer. Patients with localized melanomas can be cured by surgical excision, but only 14% of patients with metastatic melanoma survive five years due to lack of effective therapies,¹ thus underlying the importance of identifying genetic events that drive melanoma initiation and progression.

The small G-protein RAS family is frequently mutated in human solid tumors, including melanoma. Various mouse models that express oncogenic H-, K- or N-RAS specifically in melanocytes have supported the pathogenetic role of RAS proteins in melanoma. Most of these RAS-driven mouse models require additional genetic alterations and/or UV irradiation for melanoma pathogenesis, thus supporting the importance of delineating genetic interactions and synergy between RAS signaling and these lesions on melanomagenesis.

Genetic Alterations Observed in Melanoma

Genetic analyses of melanoma specimens have identified several aberrantly regulated pathways, including INK4a-CDK4/6-RB, ARF-p53-MDM2, RAS-RAF-MAPK, PTEN-PI3K-AKT and aMSH-MC1R-cAMP-MITF via genetic, genomic or epigenetic mechanisms.² The top five genes mutated in malignant melanomas, as identified by the Sanger Institute COSMIC (Catalogue Of Somatic Mutations In Cancer)³ website (www.sanger.ac.uk/cosmic), are shown in Table 1.

Table 1.

Top 5 genes mutated in malignant melanoma

Gene name	Sample number	Positive samples	Percent mutated
BRAF	5492	2269	41%
NRAS	3461	686	20%
CDKN2A	1056	281	27%
KIT	1098	89	8%
PTEN	544	85	16%

The mutation data was obtained from the Sanger Institute Catalogue Of Somatic Mutations In Cancer Web site, (http://www.sanger.ac.uk/cosmic).³⁰

The high frequency of BRAF mutations⁴ and the presence of the less common yet reciprocal NRAS mutations⁵ support the importance of the RAS-RAF-MAPK pathway in melanoma. Among the three closely related RAS proteins, NRAS is the most commonly mutated RAS family member (20%), but mutations on KRAS (2%) and HRAS (1%) have also been observed in melanoma specimens.³ The activating mutations on codon 12, 13 or 61 of H-, N- and K-RAS proteins result in constitutive RAS signaling, which activates RAF kinase family, PI3K, RalGDS and phospholipase Cϵ.⁶ The RAF kinase family, consisting of A-, B- and C-RAF, is a serine/threonine kinase that activates the MAPK pathway. BRAF is the most frequently mutated gene in human melanocytic neoplasms, with frequencies of 82% in benign nevi and 50% in melanomas.^4,7,8

Activation of the PI3K-AKT pathway is often achieved by activation of receptor tyrosine kinases, RAS mutation,^9,10 inactivation of its negative regulator PTEN (phosphatase and tensin homologue deleted on chromosome 10) or AKT activation.⁸ PTEN resides on chromosome 10q23-24, the frequently deleted region in melanoma.^11,12 Allelic loss or mutation of PTEN has been observed in 5–15% of uncultured melanoma specimens and metastasis, 17% of short-term melanoma cultures, and 30–40% of established melanoma cell lines.^5,13–15 PTEN functions as a dual lipid/protein phosphatase and hydrolyzes 3-phosphate on phosphatidylinositol-(3,4,5)-trisphosphate that promotes survival, growth and proliferation, leading to inhibition of AKT activation. When activated, AKT stimulates cell cycle progression, survival, metabolism and migration through phosphorylation of many physiological substrates. Increased AKT3 expression has been reported to accompany DNA copy gain,^16,17 whereas amplification or gain of the AKT1 or AKT2 locus has not been observed in melanoma. A rare activating mutation (E17K) was identified on AKT3 in 2 out of 65 melanoma cell lines and 2 out of 137 melanoma specimens as well as on AKT1 in one melanoma sample in the same study. E17K mutation was not observed on AKT2.¹⁸

Mouse Models of Melanoma Initiation and Progression

The pathogenetic roles of the genetic alterations identified in human melanoma specimens have been successfully elucidated in genetically engineered mouse models. Why the NRAS mutation prevails in melanoma is not yet clear; however, expression of all three activated RAS isoforms predisposes to melanoma. The melanocyte-directed HRAS^V12G transgene, combined with inactivating mutations in Ink4a, Arf or p53, promotes development of non-metastatic melanomas.^19–21 In contrast, an activated NRAS^Q61K transgene expression in melanocytic lineages in Ink4a/Arf-deficient mice drives cutaneous melanomas, as well as metastatic spread to lymph nodes and other distal sites (e.g., lung and liver) in one-third of the cases.²² KRAS^V12G expression in melanocytes utilizing Cre-recombinase/LoxP system (Tyr::CreERT2) has been shown to lead to melanoma formation with median latency of 4 months without additional genetic manipulation or exposure to UV light.23 However, somatic loss of Ink4a p16 and/or p53 in melanocytes²⁴ showed synergism with KRAS^V12G expression and enhanced melanoma formation. These genetic studies have clearly established the tumorigenic role of activated RAS signaling in melanoma and revealed similarities and dissimilarities of signaling driven by RAS isoforms.

Consistent with the frequent observation of BRAF V600E mutations in benign nevi,^4,7 BRAF^V600E expression in melanocytes induced a nevoid hyperpigmentation phenotype in mice^25–27 with rare progression to melanoma with²⁵ or without²⁶ accompanying Ink4a p16 loss. Similar to observations made in RAS-driven models, oncogenic BRAF expression in melanocytes synergistically interacts with Ink4a/Arf loss and p53 loss²⁵ or Ink4a p16 loss.²⁶ These compound models showed enhanced melanoma formation with decreased latency and increased penetrance.

Contribution of PTEN inactivation in melanoma tumorigenesis has been shown previously. Although neither Pten^+/− nor Ink/Arf^−/− mice developed melanomas, Pten heterozygosity in Ink4a/Arf null mice caused melanoma formation in a small number of mice (3/46; 6.5%) at 28–31 weeks,²⁸ supporting the cooperative interaction of dual inactivation of PTEN and INK4a/ARF. Moreover, loss of PTEN expression in mouse melanocytes synergized with activated BRAF, leading to robust melanoma formation and development of metastases,²⁷ supporting genetic interactions of the RAS/RAF/MAPK and PTEN/PI3K/AKT pathways in melanoma progression.

Genetic Interaction of HRAS Activation and PTEN Loss in Melanoma Initiation and Progression

Recently, we addressed the genetic interactions of three melanoma-prone genetic elements, namely RAS activation, PTEN loss and INK4a/ARF deficiency, utilizing genetically engineered mouse models. Our results showed evidence of cooperation among these genetic lesions, manifested as accelerated melanoma development and enhanced migration of melanoma cells. Specifically, inactivation of one copy of Pten in mice with melanocyte-directed oncogenic HRAS^V12G expression and loss of Ink4a/Arf (hereafter RAS-Ink4a/Arf¹⁹) led to an earlier onset of melanoma and decreased overall melanoma-free survival (29.6 versus 18.9 weeks in RAS-Ink/Arf mice with Pten^+/+ versus Pten^+/− genotypes, respectively). Pten^+/− Ink/Arf^−/− mice without RAS expression did not develop melanoma and succumbed to non-melanoma-related death (median survival of 19.4 weeks). In the period prior to the appearance of non-melanoma tumors, 75% of the Pten^+/− RAS-Ink4a/Arf mice developed melanoma compared with 35.7% of mice with wild-type Pten. Histopathologically, these primarily spindle cell melanomas are similar to those observed in the RAS-Ink4a/Arf model.^19,29 Moreover, we also observed 2 cases of melanoma metastasis among the 21 tumor-bearing RAS-Ink4a/Arf mice heterozygous for Pten, which was never observed in RAS-Ink4a/Arf mice. At the molecular level, RNAi-mediated Pten inactivation in RAS-Ink/Arf melanoma cells led to E-cadherin downregulation and AKT2 phosphorylation, accompanying enhanced migration and invasion compared to control cells without Pten knock-down. Both the E-cadherin downregulation and the AKT2 activation have been previously shown to promote progression of various tumors.^30–32 These data support a role for PTEN as a suppressor of melanoma progression. An inverse correlation of PTEN level to Breslow depth and melanoma progression has been consistently reported.³³ Therefore, HRAS activation, loss of PTEN and loss of INK4a/ARF cooperate to drive the genesis and progression of melanoma. Similarly, cooperation of KRAS activation and PTEN loss have been reported in mouse models of lung³⁴ and pancreatic cancer,³⁵ and HRAS activation and PTEN loss have been reported in squamous cell carcinoma model.³⁶

This synergy was unexpected since the activation of RAS and the loss of PTEN have been considered as functionally and genetically redundant events in melanoma, due to the common effects of NRAS and PTEN alterations leading to AKT activation. Moreover, the relative reciprocity of NRAS and PTEN alterations was previously proposed in melanoma specimens⁵ and cell lines.³⁷ However, in human melanoma, we found that 14% (2/14) of the NRAS-mutated tumor samples harbor PTEN loss or mutation. Conversely, 17% (2/12) of the melanoma samples with PTEN loss or mutation also contain NRAS mutation (Chin L, unpublished data). Thus, concurrent RAS activation and PTEN loss occur in melanoma, and this subtype may have a higher tendency to metastasize considering the phenotype observed in the mouse model. Schematic model for the cooperative interactions of PTEN deficiency and RAS activation in melanoma is shown in Figure 1.

Figure 1.

Schematic model for the cooperative interactions of PTEN deficiency and RAS activation. (A) Activated RAS (RAS*) causes RAF/MAPK activation and PI3K/AKT activation, increasing pAKT1 and 3 preferentially. (B) PTEN inactivation and RAS activation cooperates to increase pAKT2 along with pAKT1 and pAKT3.

Questions that Remain to be Answered

The cooperation of MAPK and AKT pathways in melanoma tumorigenesis and progression is well supported by our study³⁸ employing oncogenic HRAS and by Dankort et al.²⁷ with activated BRAF combined with Pten loss. Considering the prevalence of activating NRAS mutations in melanoma, it is important to confirm whether oncogenic NRAS also synergizes with PTEN loss in melanoma tumorigenesis. Some biological differences exist among highly homologous RAS isoforms, including lipid raft localization of HRAS and NRAS, but not KRAS,³⁹ and more potent activation of PI3K/AKT by NRAS and HRAS than by KRAS in fibroblasts and melanocytes.^40,41 However, melanomas developed in similarly designed NRAS^Q61K and HRAS^V12G driven mouse models in Ink4a/Arf null background show indistinguishable histological features and similar molecular profiles (Kwong and Chin, unpublished data). In addition, we observed that RNAi-mediated PTEN loss in human melanoma cell line WM1366 harboring NRAS^Q61L mutation recapitulated the observation made in HRAS^V12G driven mouse melanoma cells, namely, increased invasion in a Boyden chamber assay accompanied by enhanced AKT2 activation. These data suggest the similarity for the role of HRAS and NRAS activation in melanoma tumorigenesis. Mutation of BRAF, a downstream target of RAS signaling, is frequently observed with concurrent PTEN loss. In one study, 3 out of 7 melanoma specimens with reduced PTEN protein expression showed BRAF mutation, while none of the 7 showed mutation on RAS isoforms.³³ About half of the melanoma cell lines with BRAF mutation showed concurrent PTEN loss, whereas only 1 out of 11 melanoma cell lines with NRAS mutation showed PTEN loss.⁴² Collectively, all of these HRAS, NRAS and BRAF mutations share one feature, MAPK activation, which cooperatively interacts with PTEN loss in melanoma tumorigenesis.

At the molecular level, we reported increased pAKT2 and decreased E-cadherin protein levels upon PTEN inactivation in RAS-activated melanomas and cell lines. RAS proteins positively regulate AKT by directly binding to the p110 catalytic subunit of PI3K⁹ and by activating autocrine signals involving EGFR family ligands.¹⁰ In our study, mouse and human melanoma cells with activating RAS mutations showed AKT activation, and loss of PTEN expression in these cells increased phosphorylation of AKT2 isoform (Fig. 1). It is not clear whether signaling from activated RAS preferentially activates AKT1/3 and represses AKT2, which is activated by PTEN loss. In addition, AKT2 was reported as a downstream target of metabotropic glutamate receptor 1 (GRM1), of which expression in mouse melanocyte leads to melanoma formation, and loss of Akt2 in a Grm1-melanocytic cell line suppressed invasion in vitro.⁴³ However, AKT2 mutation or genomic amplification/gain has not yet been observed in melanomas. On a related note, Stahl et al. showed that RNAi-mediated PTEN loss in melanocytes and WM35 melanoma cells containing BRAF V600E mutation led to increased pAKT3, which inhibited apoptosis.¹⁶ Therefore, it will be important to address whether AKT isoforms become differentially activated in specific genetic backgrounds and whether they play a distinct role during melanoma genesis and progression. Similar to that shown for breast cancer,³² low AKT2 activity at early stage may have growth advantage, and AKT2 activation at a later stage may promote progression. Moreover, AKT2 was reported to induce the miR-200 microRNA family, which in turn decreases E-cadherin expression.⁴⁴ Therefore, it will be of interest to assess whether miR-200 is induced in our model and whether it is responsible for the observed downregulation of E-cadherin. Further systemic and comprehensive work is needed to identify the activity of each AKT isoform in melanoma tumorigenesis.

Extra View to: Nogueira C, Kim KH, Sung H, Paraiso K, Dannenberg JH, Bosenberg M, et al. Cooperative interactions of PTEN deficiency and RAS activation in melanoma metastasis. Oncogene. 29:6222–6232. doi: 10.1038/onc.2010.349.