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[Cancer Research 60, 1800-1804, April 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Relative Reciprocity of NRAS and PTEN/MMAC1 Alterations in Cutaneous Melanoma Cell Lines1

Hensin Tsao, Xue Zhang, Kianna Fowlkes and Frank G. Haluska2

Department of Dermatology (H. T.), Division of Hematology/Oncology (H. T., X. Z., K. F., F. G. H.), Massachusetts General Hospital and Dana-Farber/Partners CancerCare, Boston, Massachusetts 02114, and Department of Medical Genetics, China Medical University, Shenyang, People’s Republic of China (X. Z.)


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Both inactivation of the tumor suppressor gene, PTEN/MMAC1, and oncogenic activation of RAS have been described in human cutaneous melanoma. In mice, activation of a RAS-containing pathway is a necessary step in the pathogenesis of murine melanomas. Because PTEN negatively regulates on the downstream effects of phosphatidylinositol-3-kinase (PI3-K), we hypothesized that the loss of PTEN/MMAC1 and the activation of RAS may be largely equivalent because RAS is a known positive upstream regulator of PI3-K. We expanded our previous survey of PTEN/MMAC1 mutations and analyzed the RAS status of 53 cutaneous melanoma cell lines, 18 glioma cell lines, and 17 uncultured cutaneous melanoma metastasis. Overall, 51% of the cell lines had alterations in either PTEN/MMAC1 or RAS. We found 16 cell lines (30%) with alterations in PTEN/MMAC1 and 11 cell lines (21%) with activating NRAS mutations; only 1 cell line had concurrent alterations in both genes. Moreover, glioma cell lines with a high frequency of PTEN/MMAC1 inactivation had no identifiable RAS alterations. Ectopic expression of PTEN in several cutaneous melanoma cell lines suppressed colony formation irrespective of PTEN/MMAC1 status; furthermore, PTEN expression in cell lines carrying activated RAS also suppressed colony formation. The relative reciprocity of PTEN/MMAC1 abrogation and NRAS activation suggests that the two genetic changes, in a subset of cutaneous melanomas, are functionally overlapping.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The incidence of cutaneous melanoma has been rising over the past several decades. Although the pathogenetic mechanisms underlying melanoma tumor formation are still largely unknown, several genes have been shown to be targets for mutations in cutaneous melanoma. CDKN2A is the most frequently inactivated tumor suppressor gene in cutaneous melanoma (reviewed in 1 ), whereas the RAS genes are the most commonly mutated oncogenes described thus far for melanoma (2, 3, 4, 5, 6, 7) . Using these observations, Chin et al (8) generated a murine model of cutaneous melanoma that reflects the human genetics by melanocyte-specific expression of an oncogenic RAS gene on a CDKN2A-null background. Taken together, the human and murine data suggest that a RAS-dependent pathway is distinct from, and cooperates with, the CDKN2A/retinoblastoma (pRb) pathway in melanoma tumorigenesis. However, the downstream components of the RAS-affected pathway(s) in cutaneous melanoma are unknown.

We and others have recently reported that approximately 30% of cutaneous melanoma cell lines harbor mutations or deletions of the tumor suppressor gene, PTEN/MMAC1 (9 , 10) . Sequence analysis of the PTEN/MMAC1 gene revealed a dual serine/threonine and tyrosine phosphatase domain (11 , 12) , whereas biochemical analyses identified a lipid phosphatase function that can dephosphorylate PtdIns(3 , 4 , 5) P33 (13 , 14) . In mouse studies, homozygous elimination of PTEN/MMAC1 leads to early embryonic lethality (15, 16, 17) . Stambolic et al. (15) demonstrated that murine embryonic fibroblasts that lack PTEN function have elevated levels of PtdIns(3 , 4 , 5) P3 and PKB activity, a downstream signal target for PtdIns(3 , 4 , 5) P3. These biochemical data suggest that one function of PTEN is to negatively regulate the PI3-K/PKB pathway.

Several lines of evidence point to a possible genetic relationship between RAS and PTEN/MMAC1. Malignancies that have high rates of RAS mutations, such as colon cancer (18, 19, 20, 21) and pancreatic cancer (22) , have low rates of PTEN/MMAC1 alterations (23 , 24) ; on the other hand, gliomas have a high frequency of PTEN/MMAC1 inactivation (11 , 12 , 25, 26, 27) but low rates of RAS mutations (28 , 29) . Biochemically, the induction of PI3-K activity and intracellular levels of PtdIns(3 , 4 , 5) P3 is mediated through RAS (30, 31, 32) , and, thus, PTEN's impact on this pathway may be affected by the level of RAS activity. We hypothesized that the loss of PTEN [a negative regulator of PtdIns (3 , 4 , 5) P3 levels] and the activation of RAS (a positive regulator of PI3-K) are functionally and—potentially genetically—equivalent in at least a subset of cutaneous melanomas. We, thus, set out to assess for frequency and type of RAS mutations in our panel of melanoma specimens that have been characterized for PTEN/MMAC1 alterations.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell lines and DNA.
The human melanoma cell lines have been described previously (9) . In addition, A375, CHL-1, Malme, and HS597 melanoma cell lines were obtained from American Type Culture Collection (Rockville, MD); the K melanoma cell lines were from Dr. G. Dranoff (Dana-Farber Cancer Institute, Boston, MA). Cells were grown in DMEM media supplemented with 10% FCS and antibiotics.

DNA from 18 glioma cell lines was provided by Dr. G. Robertson (Ludwig Institute for Cancer Research, San Diego, CA). DNA samples of uncultured metastatic melanomas (33) were obtained from Dr. K. Huebner (Kimmel Cancer Center, Philadelphia, PA).

A pSG5-PTEN plasmid containing PTEN cDNA was obtained from Dr. W. Sellers (Dana-Farber Cancer Institute, Boston, MA). The insert was subcloned into the pIRESpuro vector (Clontech, Palo Alto, CA) and pCDNA3.1neo vector (Invitrogen, Carlsbad, CA).

PCR-SSCP.
Primers and conditions for PCR-SSCP analysis of the PTEN/MMAC1 gene have been described previously (9) . PCR-SSCP analysis of the RAS genes used the following primer sets: (a) HRAS Exon 1F: 5'-CAGGCCCCTGAGGAGCATG-3'. and HRAS Exon 1R: 5'-GTATTCGTCCACAAAATGGTTCT-3'; (b) HRAS Exon 2F: 5'-TCCTGCAGGATTCCTACCGG-3', and HRAS Exon 2R: 5'-GGTTCACCTGTACTGGTGGA-3'; (c) KRAS Exon 1F: 5'-GGCCTGCTGAAAATGACTGA-3', and KRAS Exon 1R: 5'-GTCCTGCACCAGTAATATGC-3'; (d) KRAS Exon 2F: 5'-TTCCTACAGGAAGCAAGTAG-3' and KRAS Exon 2R: 5'-CACAAAGAAAGCCCTCCCCA-3'; (e) NRAS Exon 1F: 5'-CAGGTTCTTGCTGGTGTGAAATGACTGAG-3', and NRAS Exon 1R: 5'-CTACCACTGGGCCTCACC-TCTATGG-3'; and (f) NRAS Exon 2F: 5'-GTTATAGATGGTGAAACCTG-3', and NRAS Exon 2R: 5'-ATACACAGAGGAAGCCTTCG-3'.

Amplification was carried out in 10-µl reaction mixtures containing 1 µl of DNA, 2 mCi [{alpha}-32P]dCTP (NEN, Boston, MA), and 1 mM each primer under standard conditions. The samples were denatured at 95°C for 5 min, annealed for 30 s using a touchdown protocol (62°C for 2 cycles, 60°C for 2 cycles, 59°C for 2 cycles, 58°C for 3 cycles, 57°C for 3 cycles, 56°C for 3 cycles, and 55°C for 15 cycles), extended at 72°C for 30 s with a final primer extension at 72°C for 10 min. The reactions were stopped with four volumes of stop buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). Samples were denatured at 95°C for 5 min, chilled on ice immediately for 5 min, and loaded directly onto a 0.5x MDE gel (FMC BioProducts, Rockland, ME), with and without glycerol, in 0.6x Tris-Borate EDTA (TBE) buffer. Fragments were subjected to electrophoresis at 4 W overnight at room temperature. After electrophoresis, the gels were dried and exposed to autoradiographic film without a screen for 12–24 h.

DNA Fragments showing mobility shifts were then prepared by PCR under the same condition, separated on agarose gel, purified using QIAquick kit (QIAGEN, Inc., Santa Clarita, CA), and directly sequenced using AmpliCycle sequencing kit (Perkin-Elmer, Foster City, CA) or submitted to the Massachusetts General Hospital Sequencing Core Facility for automated sequencing.

Colony Growth Suppression Assay.
On the night before transfection, target cells were plated at 500,000 per well in a 6-well plate with DME/10% FCS (no antibiotics). One to two µg of column-purified plasmid (QIAGEN, Inc., Santa Clarita, CA) was transfected with Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD) using the manufacturer’s protocol. After 48 h, the cells were trypsinized into 100-mm dishes and allowed to settle overnight. The cells were then selected with the appropriate concentrations of G418 or puromycin (Sigma, St. Louis, MO) for 2–4 weeks and stained with Giemsa.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Prevalence of NRAS Alterations in Cell Lines.
Using PCR-SSCP, we evaluated 53 cutaneous melanoma cell lines, 17 uncultured cutaneous melanoma metastases and 18 glioma cell lines for mutations in codons 12/13/61 of NRAS, KRAS, and HRAS. We found a total of 11 NRAS mutations (11 of 53 or 21%; 1 at codon 12; 10 at codon 61) from our melanoma cell lines and 2 NRAS codon 61 mutations from our 17 uncultured melanoma metastases (2 of 17 or 12%). We did not detect any HRAS or KRAS mutations in any melanoma samples. Fig. 1Citation is a representative PCR-SSCP gel delineating multiple NRAS exon 2 (codon 61) fragments with aberrant migration patterns. Fig. 2Citation shows the sequencing chromatograms for the NRAS codon 12 mutation (Fig. 2ACitation ) and the three NRAS Gln61 alterations (Fig. 2BCitation ). Table 1Citation lists all of the NRAS mutations from our cutaneous melanoma cell lines. Two uncultured melanoma metastases had NRAS Gln61Arg mutations.



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  		Fig. 1. Representative PCR-SSCP of NRAS Exon 2 in a set of cutaneous melanoma cell lines. On sequencing, the aberrant migrating fragments seen in Lanes 1, 7, 11, 27, and 33 harbored a NRAS Gln61Lys mutation; the aberrant migrating fragments seen in Lanes 3, 6, and 31 harbor a NRAS Gln61Arg mutation; the aberrant migrating fragment seen in Lane 19 is a Gln61Leu mutation.

 


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  		Fig. 2. Sequencing chromatograms for the NRAS Gly12 mutation (A) and NRAS Gln61 mutations (B).

 

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  		Table 1 PTEN/MMAC1 and NRAS status of cutaneous melanoma cell lines

 
We found no HRAS, KRAS, or NRAS codon 12/13/61 mutations in the 18 glioma cell lines (data not shown).

Relative Exclusivity of PTEN/MMAC1 and NRAS Alterations.
PTEN/MMAC1 was altered in 16 of our melanoma cell lines (16 of 53 or 30%; Table 1Citation ). NRAS was mutated in 1 of 16 of the melanoma cell lines with PTEN/MMAC1 mutations and 10 of 37 of the melanoma cell lines with wild-type PTEN/MMAC1. Overall 27 (51%) of 53 of our cutaneous melanoma cell lines had either PTEN/MMAC1 or NRAS mutations, although only 1 cell line (cell line HS 944) had alterations in both genes. The two uncultured cutaneous melanoma metastases that harbored NRAS codon 61 mutations had wild-type PTEN/MMAC1 (data not shown). Furthermore, Furnari et al. (34) have previously shown that 14 of the 18 glioma cell lines harbor PTEN/MMAC1 alterations.

In our total analysis, 12 of 56 specimens with normal PTEN/MMAC1 harbored NRAS mutations compared with 1 of 32 specimens with aberrant PTEN/MMAC1 (Fisher’s exact test, P = 0.027); thus, the reciprocity of mutations does not appear to be random.

Suppression of Colony Formation by PTEN in the context of PTEN/MMAC1 and RAS Genotypes.
We next explored the in vitro colony suppressive function of PTEN in the context of defined genetic backgrounds. Fig. 3ACitation shows colony suppression by PTEN in a PTEN/MMAC1 del/RAS wt background (cell line UACC903), PTEN/MMAC1wt/RASwt background (cell line A375) and PTEN/MMAC1 wt/RAS mut (cell line SK-Mel 119) background. Fig. 3BCitation shows the relative suppression by PTEN in a set of cutaneous melanoma cell lines for which the PTEN/MMAC1 status and RAS status of each line had been determined. Each bar represents an individual experiment; the light gray bars used the pCDNA3 vector whereas the dark gray bars used the pIRES vector. Exogenous introduction of PTEN into cutaneous melanoma seems to uniformly suppress colony growth independent of PTEN/MMAC1 status. Furthermore, enforced expression of PTEN is also able to suppress growth of cell lines with mutated NRAS.



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  		Fig. 3. A, growth suppression by PTEN in a PTEN/MMAC1del/RAS wt background (cell line UACC903), PTEN/MMAC1 wt/RAS wt background (cell line A375), and PTEN/MMAC1 wt/RAS mut (cell line SK-Mel 119) background. B, graphical representation of PTEN suppression among various cell lines in experiments using pCDNA3 vectors (light gray bars) and pIRES vectors (dark gray bars).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Our results suggest, for the first time, that RAS and PTEN/MMAC1 may be genetically linked in at least a subset of cutaneous melanomas and that oncogenic activation of the former and inactivation of the latter may be largely equivalent. This relative reciprocity suggests that RAS and PTEN/MMAC1 may lie on a genetic pathway that is commonly abrogated in cutaneous melanoma. Consistent with the melanoma cell line results, the glioma cell lines have a high rate of PTEN/MMAC1 inactivation (14 of 18 or 78%; Ref. 34 ) but no detectable activating RAS mutations. Two models are consistent with our observations. If the activation of RAS and the loss of PTEN/MMAC1 are equivalent, then in the setting of a mutation in one of these genes, no selective advantage would be conferred by a second mutation. In other words, in cells carrying one mutation, a second mutation would be superfluous. To date, functional data that suggest a reciprocal regulatory effect of RAS and PTEN on PI3-K support this model best. Alternatively, the presence of oncogenic RAS and concurrent PTEN/MMAC1 loss may actually be selectively disadvantageous. For instance, activated RAS can promote apoptosis in certain genetic backgrounds (35, 36, 37) , and, if RAS activation and PTEN/MMAC loss function cooperatively in this manner, one would not see concomitant mutations of both genes. Our finding of a single cell line that contains both RAS and PTEN/MMAC1 alterations may reflect the divergence of RAS pathways; in this cell line, additional RAS-mediated events may confer an even greater proliferative advantage, prevent RAS-mediated toxicity, or affect the cell at a different stage of tumor progression.

Because only one-half of our cell lines demonstrate alterations in either gene, other undetermined genes are likely involved in the remaining cell lines. Although the complete pathway integrating RAS and PTEN signaling are still unknown, several identified components are critical in cancer biology. PI3-K is known to be a downstream target of RAS (38 , 39) , and, recently, Shayesteh et al. (40) found that amplification of the PI-3 K gene, PI3KCA, may be an important step in the pathogenesis of ovarian cancers. The phospholipid products of PI-3 K, which are substrates of PTEN, activate PKB/C-AKT (41) , a protein kinase that, in its constitutively activated form (V-AKT), is a retroviral oncogene (42 , 43) . Two protein substrates of PKB are also involved in cancer: BAD (44) , a negative regulator of Bcl-2, and FKHRL1 (45) , a member of the human Forkhead family that has been shown to be involved in human malignancies (46 , 47) .

Functionally, PTEN is able to suppress growth regardless of the endogenous PTEN/MMAC1 status. Li et al. (48) reported similar findings in breast cancer. This is in sharp contrast to glioma cells, in which PTEN is ineffective in the context of a wild-type PTEN/MMAC1 (34) . This raises the possibility that other unidentified alterations are potentially upstream of PTEN in cutaneous melanoma and downstream of PTEN in gliomas. In particular, PTEN is able to suppress cell lines with normal PTEN/MMAC1 and activating NRAS mutations. The ability of PTEN to suppress cell lines with mutated RAS is consistent with a function for PTEN downstream of RAS. Along these lines, we found that PTEN is also able to suppress the growth of both NIH3T3 cells and v-RAS-transformed-NIH3T3 cells with equivalent efficacy (data not shown). In our earlier experiments, HS944 (the only cell line with both PTEN/MMAC1 and NRAS alterations) seemed relatively resistant to PTEN suppression. Some of the resistance to growth suppression that is seen in HS944 may reflect the use of the pCDNA3 plasmid instead of the pIRES vector. As can be seen for UACC903, both pCDNA3 and pIRES clearly suppressed growth, but the pIRES vector in our hands seems to be more effective. Alternatively, the intracellular levels of PtdIns(3 , 4 , 5) P3 in the presence of both activating RAS and inactivating PTEN/MMAC1 alterations may be higher than the levels resulting from either change alone, and, thus, exogenous expression of PTEN produces a stoichiometrically reduced effect.

In summary, we provide the first genetic evidence in cutaneous melanoma that PTEN/MMAC1 may be a critical component of a RAS-sensitive pathway. Both the human mutational studies and the murine models support the existence of such a pathway. Whether other genes that interact with RAS and PTEN/MMAC1 are also targeted for mutations in melanoma remains to be established. Furthermore, the robust tumor-suppressive effect resulting from restoration of PTEN may have therapeutic implications for cutaneous melanoma.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by an American Cancer Society Institutional Research Grant (to F. G. H.) and partially supported by the Marion Gardner Jackson Trust, the Warner Wellcome Research Fellowship through the Dermatology Foundation, and a Clinical Research Training Grant (CRTG-99-249-01 CCE) through the American Cancer Society (to H. T.). Back

2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 724-7081; Fax: (617) 726-6974; E-mail: haluska.frank{at}mgh.harvard.edu Back

3 The abbreviations used are: PtdIns(3, 4, 5)P3, phosphatidylinositol-3,4,5-triphosphates; PI3-K, phosphatidylinositol-3-kinase; PKB, protein kinase B; SSCP, single-strand conformation polymorphism. Back

Received 1/18/99. Accepted 2/16/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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