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. 2006 Jul;8(7):543–550. doi: 10.1593/neo.05655

Loss of Adhesion in the Circulation Converts Amelanotic Metastatic Melanoma Cells to Melanotic by Inhibition of AKT1

Zhang Weihua 1, Rachel Tsan 1, Do-Hyun Nam 1, Weixin Lu 1, Isaiah J Fidler 1
PMCID: PMC1601939  PMID: 16867216

Abstract

Direct injection of murine K-1735 melanoma cells into the subcutis, lung, or brain of syngeneic mice produces amelanotic tumors, whereas intravenous injection into the lateral tail vein or internal carotid artery produces both amelanotic and melanotic foci in the lung and the brain respectively. We hypothesized that loss of adhesion in the circulation may contribute to the melanogenic phenotypes of cells. To test this, we used enforced suspension culture of K-1735 cells by consistent rotating culture of K-1735 cells. We found that the expression of the microphthalmia transcription factor (MITF) and melanin-stimulating hormone receptor (MSHR) were upregulated in cells growing in suspension and were accompanied by inhibitions of AKT and ERK, which were reversed in cells upon regrowth as an adherent monolayer. Inhibition of the AKT pathway was responsible for MITF induction by suspension culture. Stable expression of constitutively active AKT significantly repressed the melanogenesis of K-1735 cells injected via circulation. An amelanotic clone of K-1735 cells was resistant to suspension culture-induced MITF, although the inhibition of AKT pathway was intact. Collectively, these data suggest that the inhibition of AKT pathway due to loss of adhesion within the circulation renders a subpopulation of K-1735 cells to produce melanin.

Keywords: Adhesion, circulation, melanoma, organ microenvironment, AKT

Introduction

To establish distant metastases, cancer cells must complete a series of sequential and selective steps, which includes detachment from the primary tumor, invasion and intravasation into the circulation (lymphatic or hematogenous), survival in the circulation, extravasation into the stroma of different organs, production of vasculature, and growth. The organ microenvironment can profoundly influence the biologic behavior of tumor cells, including the production of degradation enzymes, angiogenesis, resistance to chemotherapy, and induction of terminal differentiation [1]. Models of murine melanoma metastasis to the lung [2] and brain [3,4] have demonstrated multiple tumor-host interactions during the pathogenesis of metastasis. In these models, K-1735 murine melanoma cells are injected intravenously into the tail vein to produce lung metastasis, or into the internal carotid artery to produce brain metastasis in syngeneic mice. K-1735 tumors growing subcutaneously are amelanotic, whereas lung and brain metastases are a mixture of both amelanotic and melanotic lesions [5]. In a previous study, we found that genes involved in melanogenesis, such as melanin-stimulating hormone receptor (MSHR) and tyrosinase, were induced in brain metastases of K-1735 cells, but not in subcutaneous K-1735 tumors [6]; these observations suggested that the organ microenvironment is crucial to the melanogenic phenotype of K-1735 cells.

Microphthalmia transcription factor (MITF) is central to signaling pathways that control the survival, proliferation, and differentiation of melanocytes. Genes involved in melanin synthesis, such as tyrosinase [7], tyrosinase-related protein 1 [8], and MSHR [9], are regulated by MITF. Transcription factors, such as Pax3, Sox10, CREB, and Lef1, are involved in regulating the mRNA levels of MITF. Signaling pathways, such as Ras/ERK and PI3 kinase/AKT pathways, are known to be involved in the phosphorylation MITF, which facilitates the degradation of MITF at the protein level (for a review, see Goding [10]). Inhibition of AKT or ERK results in the increase of melanin synthesis in human melanocytes [11]. AKT and ERK are protein kinases that play an important role in the signaling pathway by responding to growth factors and other extracellular stimuli that regulate several cellular functions, including nutrient metabolism, cell growth, apoptosis, and survival. Many signaling pathways, including AKT and ERK, are known to be inhibited by loss of adhesion [12,13], and one of the major alterations of circulating tumor cells is loss of adherence to neighboring cells and basal membrane, loss of adhesion, and inhibitory effects of AKT and ERK on MITF protein level. This leads to the hypothesis that melanogenic phenotype may be activated in circulating melanoma cells.

To test this hypothesis, we compared melanogeneses by murine K-1735 melanoma cells introduced into organs by the circulation or by direct injection. The molecular mechanisms that regulate melanogenesis in K-1735 melanoma cells were also explored by culturing the cells in suspension. Collectively, our data suggest that loss of adhesion of circulating melanoma cells can alter their phenotype.

Materials and Methods

Reagents and Cell Lines

The K-1735 murine melanoma cell line was a gift from Dr. Margaret L. Kripke (The University of Texas M. D. Anderson Cancer Center, Houston, TX) [14]. The amelanotic C2 clone of K-1735 cells and the melanotic C4 clone were isolated as described previously [5]. Stable melanotic B16-BL6 melanoma cells isolated from the B16-F1 line syngeneic to the C57BL/6 mouse [15] served as positive control. Green fluorescent protein (GFP)-labeled K-1735 cell was prepared previously [16]. AKT inhibitor LY294002 and ERK inhibitor PD98095 were purchased from Sigma (St. Louis, MO). Polyclonal antibodies against MITF (cat. sc-10999), and β-actin (cat. sc-1616) and horseradish peroxidase-labeled secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against total AKT (tAKT; cat. 9277), phosphorylated AKT (pAKT; cat. 9271L), total ERK1/2 (cat. 9102), and phosphorylated ERK1/2 (cat. 9101L) were from Cell Signaling (Beverly, MA). Anti-CD31 antibody (cat. 557355) was from BD Biosciences (San Jose, CA). The transfection reagent Fugen6 was from Roche (Basel, Switzerland). Geneticin was purchased from Invitrogen (Carlsbad, CA).

Animals

Specific pathogen-free mice of the inbred strains C3H/HeN were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care, in accordance with current regulations and standards of the US Department of Agriculture, the US Department of Health and Human Services, and the National Institutes of Health.

Tumor Cell Injections

To produce tumors, K-1735 cells were harvested from subconfluent adherent cultures by a short treatment with 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with a medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium and then suspended in Hank's balanced salt solution. Only single-cell suspensions with > 90% viability were used for injections. Tumor cells (1.0 x 105 in 100 µl) were injected into the subcutis, tail vein, or internal carotid artery, as described previously [3]. For direct intraorgan injection, cells (1.0 x 105 in 50 µl) were injected into the left lung or into the brain (through the right parietal bone) using a 1-ml syringe with a 30-gauge needle. Tissues from animals (n = 5 in each group) were collected 3 weeks after the injections. In parallel, to track the changes of pAKT after injection through the circulation, green fluorescence-labeled K-1735 cells were used and tissues were collected and immediately frozen with liquid nitrogen at 6 hours after injection for immunohistochemical staining of pAKT and CD31.

Immunohistochemical Staining for pAKT and CD31 on Sequential Sections

Sequential frozen sections (15 µm thickness) of tissues containing GFP-labeled K-1735 cells were made with Cryostat LEICA CM3050 (Bannockburn, IL). Slides were then air-dried, fixed in cold acetone and ethanol for 2 minutes each, then air-dried before staining. The slides were blocked with a blocking solution (3% bovine serum albumin in PBS containing 0.1 % NP-40) for 1 hour at room temperature. After blocking, slides were incubated with a primary antibody against pAKT (1:100) that was diluted with blocking buffer for 3 hours at room temperature. After washing thrice in PBS (2 minutes each), the slides were incubated for 1 hour at room temperature with a secondary antibody conjugated with red fluorescence (Alexa 594). After three washes with PBS (5 minutes each), the slides were stained with fluorescent nuclear staining dye (Hoechst 33342, Molecular Probes, Eugene, OR) and mounted with antifading medium. Stained sections were examined under a Nikon Microphot-FX (Melville, NY) microscope equipped with a three-chip charge-coupled device color video camera (Model DXC990; Sony Corp., Tokyo, Japan). After photography, the pictured areas were marked on the slides. Sequential sections of pAKT-stained samples were then stained with a primary antibody against CD31 for vascular endothelial cells. Red fluorescence secondary antibody (Alexa 594) was used to visualize CD31 signal, as described above.

Cell Culture

For adherent cultures, K-1735 cells (5.0 x 106) were incubated in 100-mm plastic dishes (Corning, Corning, NY) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, l-glutamine, and two-fold vitamin solution (Gibco, Grand Island, NY) and in 5% CO2-95% air at 37°C. For enforced suspension cultures, the cells were harvested from adherent cultures (as described in the Immunohistochemical Staining for pAKT and CD31 on Sequential Sections section), incubated in 100-mm plastic dishes, and placed on a consistent rotator (120 rpm) in a CO2 incubator. To examine the properties of cells growing in suspension and then as adherent monolayers, K-1735 cells were grown for 6 hours in a dish placed on a horizontal rotator, and then the dish was removed from the rotator and the cells were allowed to grow as adherent monolayers for another 6 hours. To inhibit the activation of AKT or ERK, adherent cells that had reached 80% confluency were treated with LY294002 (10 nM) or PD98095 (25 nM) for 12 hours. Control cells were treated with a medium containing the same volume of the vehicle, dimethyl sulfoxide. There were triplicates of cell culture in each group of experiments.

Transfection of K-1735 Cells with Constitutively Active AKT

Plasmids of constitutively active AKT1 (Myc-his tagged myr-AKT1) and control vector were purchased from Upstate Biotechnology (Lake Placid, NY). myr-AKT1 and control plasmids were transfected into cells with the transfection reagent, Fugen6. Stable clones with myr-AKT1 or control vector were established by culturing transfected cells for 3 weeks in a medium with 1 mg/ml geneticin. Clones were selected by Western blot analysis, with anti-myc antibody recognizing the myc tag of myr-AKT1.

Histopathological Analysis

Tissues with tumors were collected from anesthetized mice, placed into Bouin's fluid, embedded in paraffin, and sectioned for staining with hematoxylin and eosin (H&E) for morphologic evaluation. Photographs of the organs (with tumors) were taken before and after fixation in Bouin's fluid.

Northern Blot and Western Blot Analyses

The expression level of MSHR mRNA was measured by Northern blot analysis, as described previously [4]. Western blot analysis was carried out according to protocols described previously [17]. Briefly, whole cell lysates were prepared by incubating K-1735 cells for 10 minutes at 0°C in a lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) and by centrifuging at 900g for 10 minutes, and then the supernatant was collected. To isolate membranes, the cells in a lysis buffer were disrupted by sonication (5 seconds x 5 bursts) at 0°C using a Sonic Dismambrator (Fisher Scientific, Pittsburgh, PA). The homogenate was centrifuged (900g for 10 minutes) to remove unbroken cells and nuclei, and the supernatant was centrifuged for 1 hour at 100,000g. The pellet containing membranes was suspended at a concentration equivalent to 1.0 x 106 cells/ml in the same buffer. Equal amounts of proteins were separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transblotted to nitrocellulose, blocked with 5% nonfat dry milk for 2 hours at room temperature, and incubated overnight with primary antibodies. Immunoreactive signals were visualized by enhanced chemiluminescence.

Quantification Blot Signals

The density of individual bands on each blot was quantified with a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). The band density of target molecules was normalized against the loading control band, and its fold difference, compared with the band of a control sample, was calculated by the formula: Band density of experiment/Band density of control. The fold difference that was either > 1.5 or < 0.5 was considered as significant.

Results

Intravascular Injections Produce Both Melanotic and Amelanotic Tumors

In C3H/Hen mice that received direct injections of K-1735 cells into the subcutis, lung, or brain, none of the resulting tumors was melanotic (Figure 1). In contrast, tumor foci (experimental metastases) produced in the lung subsequent to intravenous injection into the lateral tail vein were either melanotic or amelanotic. Tumor lesions produced in the brain subsequent to injection into the internal carotid artery were mostly melanotic, with some amelanotic regions.

Figure 1.

Figure 1

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Induction of melanogenesis in circulating K-1735 cells. Tumors produced by K-1735 cells injected directly into the subcutis subcutaneously (s.c.), lung intrapleural (i.p.), or brain intracerebroventricularly (i.c.) are amelanotic. In contrast, K-1735 cells introduced into the lung by injection into the lateral tail vein intravenously (i.v.), or into the brain by injection into the internal carotid artery (i.c.a.) produce melanotic lesions (bar = 10 mm). The heterogeneity of melanogenesis in brain lesions is shown by the H&E-stained ICA section. Bar = 20 µm.

Inhibition of AKT Activity in Circulating Tumor Cells

Tumor cells directly injected into tissues are likely to immediately contact the tissue stroma, whereas tumor cells injected into the circulation have a period time of suspension in the blood. Loss of adhesion usually leads to inhibition of AKT [18], and inhibition of AKT results in increased melanin synthesis [11]. We tested whether the AKT pathway was inhibited in GFP-labeled K-1735 cells that were injected into either the subcutis, the lateral tail vein, or the internal carotid artery of the same mouse (n = 5). Tissues were harvested at 6 hours after injection by immediately freezing with liquid nitrogen. As shown in Figure 2, tumor cells in the subcutis express pAKT, whereas tumor cells within the vessels do not.

Figure 2.

Figure 2

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Inhibition of AKT in circulating tumor cells. Sequential frozen sections of the brain (K-1735 cells injected through the internal carotid artery), lung (K-1735 cells injected intravenously through the tail vein), and the subcutis (subcutaneously) containing GFP-labeled K-1735 cells (arrowheads, green) were stained for pAKT (arrowhead, red), CD31 (arrow, red), and cell nuclei (Hoechst, blue). Note that the K-1735 melanoma cells within the vasculature are negative for pAKT and that the tumor cells in the subcutis are positive. Bar = 50 µm.

Melanogenesis-Related Genes Are Induced by Culturing in Suspension and Are Accompanied by Inhibition of the AKT and ERK Pathways

We test the possibility of loss adhesion contributing to melanin synthesis. We culture K-1735 cells (adherent in nature) in suspension by an enforced suspension culture using a consistent rotator. The expression of MITF and MSHR and the activation of AKT and ERK pathways were compared between cells growing in suspension and cells growing as adherent monolayers. Western blot analysis showed that, compared with adherence, growth in suspension was associated with the upregulation of MITF and the inhibition of the AKT and ERK pathways. Other than G1 arrest, the enforced suspension culture did not lead to cell death (data not shown), which is probably due to the upregulation of Bcl-2 by suspension culture (Figure 3A). Northern blot analysis revealed that the mRNA of MSHR, one of the MITF target genes for melanogenesis, was also upregulated in cells growing in suspension (Figure 3B).

Figure 3.

Figure 3

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Suspension culture upregulation of melanogenesis-related genes inhibits the AKT and ERKpathways. (A) Western blot analysis of pAKT, tAKT, phosphorylated ERK (pERK1/2), total ERK (tERK1/2), and MITF. K-1735 cells growing in suspension culture (Sp) for 3 hours exhibit upregulated MITF expression and inhibited pAKT and pERK1/2 expression. These changes are not found in adherent K-1735 cells (Ad). The target gene of MITF, Bcl-2, was also upregulated accordingly. Actin was used as loading control. Cell lysate from B16-F10 melanoma cells served as positive control. (B) Northern blot analysis of MSHR mRNA expression in K-1735 cells grown in suspension or in adherent monolayers. K-1735 cells growing in suspension culture exhibit upregulated MSHR expression. Tubulin was used as loading control. Total mRNA from B16-F10 melanoma cells served as positive control.

AKT Inhibition Is Responsible for MITF Induction

To test the specificity of AKT and ERK pathway in the upregulation of MITF, we treated adherent K-1735 cells for 12 hours with the AKT inhibitor LY294002 or the ERK inhibitor PD98095. AKT inhibition induced the expression of MITF (Figure 4A), whereas ERK inhibition did not (Figure 4B). The inhibition of AKT activation and the induction of MITF in suspended cells were reversed once the suspended cells were allowed to readhere and grow for 6 hours (Figure 5).

Figure 4.

Figure 4

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Western blot analysis of MITF expression in adherent K-1735 cells treated with AKT inhibitor LY294002 or ERK inhibitor and PD98095. (A) The AKT inhibitor LY294002 inhibited AKT phosphoryla tion and induced MITF expression without affecting levels of either tAKT or phosphorylated ERK1/2 (pERK1/2). (B) The ERK inhibitor PD98095 did not induce MITF expression, but significantly inhibited pERK1/2. Actin was used as loading control.

Figure 5.

Figure 5

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Suspension culture leads to reversible upregulation of melanogenesis-related genes. Western blot analysis shows that the inhibition of pAKT and the upregulation of MITF in suspended cells can be reversed by allowing the suspended cells to readhere for 6 hours. Actin was used as loading control.

K-1735 Cells That Express Constitutively Active AKT Do Not Produce Melanotic Lesions

To determine whether the inhibition of AKT leads to the induction of melanogenesis in K-1735 cells growing in vivo, we developed K-1735 cells to express constitutively active AKT (myc-tagged myr-AKT1). As shown in Figure 6A the expression of myc-tagged myr-cAKT1 in stably transfected cells was confirmed by the presence of myc tag recognized by anti-myc antibody, and the membrane-bound pAKT was detected in the membrane fraction of cells. K-1735 cells transfected with a control vector had significantly inhibited membrane AKT phosphorylation after 6 hours in suspension culture. In contrast, myr-cAKT1-expressing cells are resistant to the inhibition of membrane-bound AKT phosphorylation by enforced suspension culture. β-Actin from an equal volume of cytosolic fraction of each sample was used as loading control.

Figure 6.

Figure 6

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Constitutive expression of activated AKT can inhibit melanogenesis in circulating K-1735 cells. (A) Detection of membrane-bound pAKT (m-pAKT) expression by Western blot analysis in vector control cells and constitutively active AKT (myr-AKT1)-expressing cells grown in an adherent monolayer (Ad) or in suspension culture (Sp). Culturing cells in suspension for 6 hours significantly inhibits the phosphorylation of AKT in control cells, but not in myr-AKT1-expressing cells. (B) Gross anatomy of lungs with foci produced by control cells shows foci heterogeneous for melanogenesis. The foci in lungs from mice injected with myr-AKT1-expressing cells are amelanotic. (C) Gross anatomy of brain with control cells or myr-AKT1-expessing cells injected into the internal carotid artery. The brain with myr-AKT1-expressing tumor cell foci have melanin less than that of the foci produced by control cells. (D) Histologic analysis of brain tissues with foci produced by control and myr-AKT1-expressing cells. Heterogeneous expression of melanin is visible in sections containing control tumor cells. Tumor cells expressing myr-AKT1 are amelanotic. Bar = 50 µm.

K-1735 cells transfected with myr-AKT or vector control were injected intravenously into the lateral tail vein. Tumor foci in the lung of C3H/HeN mice produced by control cells were mostly pigmented, whereas foci produced by the cells transfected with myr-AKT1 were not (Figure 6B). Intravenous injection of these cells into the internal carotid artery produced tumor lesions in the brain of syngeneic mice (Figure 6C). Histologic examination (Figure 6D) revealed that control cells produced melanotic lesions, whereas the cells that expressed myr-AKT did not.

Growth in the Suspension of Amelanotic K-1735 Clone Inhibits AKT Phosphorylation But Does Not Upregulate MITF Expression

Because not all the K-1735 cells injected intravenously produce melanotic lung metastases, we examined the properties of two clones isolated previously in our laboratory [5]. Clone 2 is highly metastatic and amelanotic, and clone 4 is highly metastatic and melanotic. Clones 2 and 4 cells were grown in suspension culture. As shown in Figure 7, the phosphorylation of AKT was inhibited in both clones 2 and 4 cells; however, MITF expression was upregulated only in clone 4 (melanotic) cells grown in suspension.

Figure 7.

Figure 7

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Amelanotic C2 clone of K-1735 is resistant to suspension culture-induced melanogenic signaling, as revealed by Western blot analysis. Both the melanotic clone (C4) and the amelanotic clone (C2) respond to suspension culture (Sp) compared with adherent culture (Ad) by inhibiting the expression ofpAKT without altering the level of tAKT. However, unlike the C4 clone, in the C2 clone, MITF expression is not upregulated by growth in suspension. Actin was used as loading control.

Discussion

The interaction of cancer cells with factors unique to specific organ microenvironments determines the outcome of cancer metastasis [19]. Whether the circulation per se alters the phenotype of metastatic cells remained unclear. Tumor cells growing in organs are closely attached to each other and to host cells, and adherent culture method has been widely used for in vitro studies. Circulating tumor cells are more akin to cells growing in suspension, at least on one aspect (loss of adhesion). The loss of adhesion of circulating tumor cells can lead to multiple changes, such as the inhibition of AKT and ERK phosphorylation, thereby leading to cell death (so-called anoikis) [20]. Indeed, the majority of circulating tumor cells rapidly die [21,22], and circulating cells that are resistant to anoikis present with activated AKT, ERK, or both [23,24]. In contrast, in our study, the expression levels of pAKT and ERK in K-1735 cells growing in suspension culture were reduced, but the cells did not undergo anoikis (data not shown); indeed, the level of the antiapoptotic molecule Bcl-2 was upregulated. That Bcl-2 upregulation was accompanied by the inhibition of AKT and ERK activity is actually expected for melanoma cells because MITF, which is unique to melanocytes [10,25], can induce Bcl-2 expression [26]. Both AKT and ERK pathways are involved in the regulation of MITF [27,28]. The basal level of MITF in adherent K-1735 cells is barely detectable, which explains their amelanotic phenotype. Because the basal level of MITF in K-1735 cells is very low, the induction of MITF by the inhibition of AKT, but not ERK, suggests that the repression of MITF by AKT is likely to be on the transcription level and that the repression of MITF by ERK is likely to be on the protein level. Indeed, it has been reported that the activation of ERK facilitates the degradation of MITF through the ubiquitin-mediated proteasome pathway [29].

We found that intravenous injection of K-1735 cells into the lateral tail vein leads to the production of melanotic metastases in the lung and that intravenous injection of K-1735 cells into the internal carotid artery leads to the production of melanotic lesions in the brain. The lung or brain microenvironment per se cannot be the regulators of melanogenesis in these cells because the direct injection of K-1735 cells into the organs did not result in melanotic lesions. However, these organ microenvironments must contribute to the stabilization of melanogenesis in cells that have extravasated from the circulation and have proliferated in the organ parenchyma. This conclusion is based on the finding that the inhibition of AKT in cells within the vasculature and the induction of MITF in K-1735 cells growing in suspension culture were reversed once these cells were allowed to grow as adherent monolayers. The heterogeneity of the parental K-1735 cells for response to growth in suspension correlates with the heterogeneous melanogenic phenotype in vivo. Specifically, stable amelanotic cells (e.g., clone 2 cells) have defects in signaling that are associated with AKT inhibition and MITF induction.

The relevance of circulating tumor cells to the prognosis and therapy of cancer is gaining increased attention. A large body of research suggests that circulating tumor cells can be studied to monitor the efficacy of therapy, but little is known about the phenotypic variability of circulating cells and how the circulatory system per se affects these phenotypes [30,31]. Because all neoplasms are biologically heterogeneous [32,33], some characteristics associated with circulating cells could be associated with metastatic subpopulations [34]. Our present data suggest that phenotypes unique to circulating cells may also be due to the influence of the circulatory system. In other words, the reality of the interaction between the “seed and the soil” [35] should be extended to tumor cells in the circulation.

Acknowledgements

We thank Elizabeth Hess for critical editorial review, and Arminda Martinez for expert assistance with the preparation of this article.

Abbreviations

MITF

microphthalmia transcription factor

MSHR

melanin-stimulating hormone receptor

H&E

hematoxylin and eosin

Footnotes

1

This work was supported, in part, by Cancer Center Support Core grant CA16672 and SPORE in Prostate Cancer grant CA90270 from the National Cancer Institute, the National Institutes of Health. Z.W. was supported by the Odyssey Fellowship Award from The University of Texas M. D. Anderson Cancer Center.

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