Potential for Cripto-1 in defining stem cell-like characteristics in human malignant melanoma

Abstract

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal co-receptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.

Introduction

Human melanoma is the most rapidly increasing malignant skin disease in Caucasians ¹. Frequent exposure to ultraviolet radiation from the sun due to increased outdoor activity seems to be playing an important role in the epidemiologic increase in the incidence of melanoma ^{1, 2}. The different phases during the progression of melanoma can be summarized as radial-growth phase (RGP), vertical growth phase (VGP) and metastatic melanoma ³. Although early diagnosis and surgical excision of the primary lesion can result in high cure rates, the progression and metastatic spread of malignant melanoma to regional lymph nodes and distant organs can drastically reduce survival to months ⁴. The thickness of the lesion and involvement of lymph nodes or distant sites will determine the stage of the disease ⁵. Melanoma stages I and II are characterized by lesions less than 1mm in thickness to those greater than 4 mm in thickness but without lymph node involvement or distant metastasis. Stages III and IV can be of any thickness but will have either lymph node involvement only (Stage III) or distant metastasis with or without lymph node involvement (Stage IV). Staging of melanoma is important since this will often dictate therapeutic options and prognosis. For instance, high dose interferon alpha-2b is often chosen as an adjunct to surgical removal of non-metastatic melanomas and has been shown to improve relapse free survival ⁶. Unfortunately, treatment with specific drugs alone or in combination have not shown any significant survival advantage for patients with advanced stage or metastatic melanoma ⁷. Studies aimed at dissecting the molecular pathways involved in promoting growth, metastasis and drug resistance in malignant melanoma are imperative for the proper design and success of therapy. It has been suggested that in malignant melanoma the presence of cells that express molecules and activate signaling pathways characteristic of stem cells may be responsible for the plasticity and aggressive phenotype of this malignancy ^{8, 9}. For instance, metastatic melanoma cells express endothelial cell-associated molecules like VE-cadherin and ephrin receptor A2, highlighting the plasticity of these cells, which contributes to the formation of a paravascular perfusion pathway through a process known as vasculogenic mimicry ^{10, 11}. We have recently demonstrated that malignant melanoma cells aberrantly express the embryonic morphogen Nodal, but not its inhibitor, Lefty ¹². However, it is possible to reprogram the melanoma tumorigenic phenotype by treating these cells with human embryonic stem cell (hESC) derived Lefty and other factors. Here we discuss preliminary findings suggesting that it may be possible to exploit the expression of the stem cell marker and Nodal coreceptor, Cripto-1, to isolate cells from metastatic melanoma which appear to possess stem cell-like characteristics. In the future, the ability to identify and target these putative MSC may improve success of treatment and help reduce the risks of recurrence and metastatic spread of melanoma.

Nodal signaling in melanoma

Members of the transforming growth factor-beta family such as Nodal, Activin, GDF-1 and -3 are known to act as important morphogens and mediators of cell fate in both embryological and adult systems ¹³. Inhibition of the Nodal signaling pathway, through pharmacological inhibition of its receptor, results in hESC differentiation demonstrating the important role played by Nodal during maintenance of pluripotency of embryonic stem cells ¹⁴. Nodal has been shown to be involved during axis formation of the embryo ^15–17. Studies have shown restriction of Nodal to the left side of the Node during somatogenesis, and by the 3–4 somite stage, Nodal is confined to a subpopulation of mesoderm cells in an area which will develop into the left side of the embryo ¹⁸. The asymmetric patterning of Nodal expression is detected through the stages of normal heart and axis development until the 12–14 somite stage, after which Nodal expression is significantly reduced ^{18, 19}. Observations in zebrafish have shown that specification of the anterior axial mesoderm is also induced by high levels of Nodal ²⁰.

In recent studies we have shown that aggressive VGP melanoma and melanoma metastases express Nodal whereas it was not detected in normal skin or in normal melanocytes, and was absent in non-invasive RGP melanomas ²¹. This expression not only correlated with melanoma progression it was also found to be important for maintaining tumor plasticity. In fact, treatment of Nodal expressing melanoma cells with a synthetic inhibitor of the receptor complex Alk 4/5/7, important for Nodal signaling, or with an anti-Nodal specific morpholino significantly reduced invasiveness and tumor growth, reduced the expression of plasticity markers, such as VE-cadherin and Keratin 18, and caused these cells to re-express Tyrosinase, a marker for melanocyte differentiation ²¹. These findings suggest that Nodal signaling may represent a common link for the convergence of embryonic and tumorigenic signaling pathways in malignant melanoma and implicate Nodal as a marker for disease progression and a possible new therapeutic target.

Epigenetic reprogramming of aggressive melanoma cells

Given the undifferentiated molecular signature of aggressive melanoma cells ^{10, 11}, we explored the possibility of reprogramming these tumor cells to a less aggressive phenotype by exposing them to embryonic microenvironments. In one study, GFP-labeled human metastatic melanoma cells did not form tumors and assumed neural crest-like characteristics participating in the formation of neural crest derived structures like branchial arches, dorsal root and sympathetic ganglia when transplanted in ovo in proximity to embryonic chick neural crest cells ²². These results demonstrate the ability of a subset of melanoma cells capable of responding to embryonic environmental cues to undergo reprogramming of their metastatic phenotype.

A 3-D model was also developed to study the capacity of hESC-derived factors to affect the tumorigenic phenotype of aggressive melanoma cells ²³. In this system, hESCs are seeded onto a 3-D matrix for up to 4 days and then removed, leaving a conditioned 3-D matrix onto which human metastatic melanoma cells are seeded and grown for 3–4 days. Under these conditions, the melanoma cells were induced to form spheroids, express the melanocyte marker MLANA, and demonstrated significantly reduced invasive behavior in vitro and tumor growth potential in vivo ²⁴. These results indicate that factors derived from the hESCs and deposited in the 3-D matrix are capable of reprogramming the metastatic potential of melanoma cells. Since Nodal has been shown to be an important growth factor for aggressive melanoma cells in vitro, it is reasonable to suspect that Nodal inhibitors such as Lefty, which can regulate Nodal expression and/or signaling ²⁵, may represent a possible candidate capable of affecting the metastatic phenotype of melanoma. In fact, Lefty was expressed by hESCs but not by metastatic melanoma cells and was detected in the hESC-conditioned 3-D matrix onto which the Nodal expressing melanoma cells were subsequently seeded and found to lose their aggressive phenotype ¹². This study showed that the ability to suppress the tumorigenic phenotype of the melanoma cells was directly associated with the hESC-derived Lefty, since other human stem cell types such as those derived from amniotic fluid, umbilical cord blood, adult bone marrow or embryo-associated human cytotrophoblast cells did not express significant levels of Lefty. In addition, further confirmation of Lefty’s effect on the metastatic melanoma cells was obtained when the 3-D matrix was conditioned by hESCs treated with anti-Lefty morpholinos; this failed to inhibit Nodal expression in melanoma cells or change their metastatic phenotype ¹². However, it cannot be excluded that other Nodal signaling partners such as Cripto-1 can also be affected by Lefty. In fact, Lefty, in addition to restricting Nodal binding and signaling levels ²⁵, can also bind to Cripto-1 thus preventing Nodal from forming a more active signaling complex with the type I and type II Activin receptors ²⁵. Interestingly, only a minor subpopulation of melanoma cells were found to express Cripto-1¹² and future studies will determine whether this small fraction of Cripto-1-positive cells play a significant role in the context of Nodal signaling in metastatic melanoma. Additionally, Tomoregulin (TMEFF1), a transmembrane protein containing two follistatin domains and an EGF-motif, has been shown to inhibit Nodal signaling in early Xenopus embryos by binding to the CFC domain of Cripto-1 and sequestering it from the ALK4 receptor, thus preventing Cripto-1 from functioning as a coreceptor for Nodal ²⁶. However, TMEFF1 does not appear to play a significant role in inhibiting Nodal signaling by hESCs, as Western blot analysis did not detect significant levels of TMEFF1 in hESC conditioned matrix, but revealed TMEFF1 expression by melanoma cells (Figure 1). Finally, Cerberus, a member of the cysteine-knot superfamily which includes DAN and Gremlin ²⁷, is known to directly bind and block Nodal signaling ²⁸. Since Cerberus can be detected in hESC conditioned matrix and not in melanoma cells (Figure 1), future studies will need to determine to what extent Cerberus can affect Nodal signaling in metastatic melanoma cells and whether these effects are additive or synergistic to those observed with Lefty.

Figure 1.

Expression of potential Nodal signaling antagonists in human embryonic stem cell-conditioned matrix (H9CMTX) and human melanoma cell lines (A375M, C8161, WM278). Results from Western blot show expression of Tomoregulin (TMEFF1) in cell lysates from A375M, C8161 and WM278, whereas TMEFF1 was not detected in H9CMTX. Cereberus was detected by Western blot in H9CMTX, but not detected in any of the melanoma cell lines analyzed.

Stem cell-like subpopulation in melanoma

Just as normal somatic stem cells contained in specialized compartments or niches are responsible for the growth, maintenance and repair of the tissue in which they reside, it is becoming an increasingly popular notion that cancer stem cells, in a similar way, may be responsible for some of the characteristics of the primary tumor in which they are found. Normal stem cells have increased capacity for self-renewal through activation of signaling pathways such as Wnt, Notch and Hedgehog ²⁹. They also show increased activity of pro-survival pathways including PI3K/Akt and MAPK/ERK signaling pathways ³⁰ and high expression levels of ATP-binding cassette (ABC) transporters which give these cells their characteristic resistance to drugs and toxins ³¹. Slowly proliferating, non-pigmented, undifferentiated melanocyte stem cells with the capacity for self renewal have been identified ³². Thus, it is likely that melanoma lesions may contain melanoma stem cells. For instance, malignant melanomas have been shown to contain cells that express factors associated with normal stem cells, such as MDR1, also referred to as P-glycoprotein, responsible for multidrug-resistance ³³. It is thought that this subpopulation of undifferentiated, self-renewing, drug resistant cells may be responsible for the failure of chemotherapy, disease recurrence and metastatic spread. Strategies for identifying and targeting these potential MSC could improve the efficacy of current therapies and reduce the rates of recurrence and metastasis. Different reports have described ways of potentially isolating MSC. Subpopulations of metastatic melanoma cells have been isolated on the basis of the expression of molecules characteristic of pluripotent stem cells ¹¹. For instance, the expression of the drug efflux transporter ABCG2 and the stem cell marker CD133 has been proposed to be useful for identifying a putative MSC population ³⁴. Serial in vivo transplant experiments will eventually determine whether these ABCG2⁺/CD133⁺ melanoma cells retain the capacity for self-renewal and differentiation characteristic of cancer stem cells. More recently, MDR1 was used to FACS sort and isolate melanoma cells that showed features common to stem cells ³⁵. In that study, MDR1-positive malignant melanoma cells showed increased capacity for clonogenicity and self renewal and expressed the stem cell related transcription factor Nanog.

It has also been demonstrated that a subpopulation of melanoma cells can form non-adherent spheres when grown in media used to culture hESCs ^{24, 36}. These cells were shown to differentiate into melanocyte, adipocyte, osteocyte, and chondrocyte lineages and to express CD20 ³⁶. The self-renewal characteristics of these cells was demonstrated by persistence of the sphere-forming capacity after multiple passages in vitro and after re-plating cells from tumors that formed in xenograft models. Over time, however, these cells eventually lost their capacity to differentiate suggesting that there are limitations to the sphere forming assay for enrichment of MSC.

Using a similar strategy, we have attempted to isolate a MSC population by separating the potential MSC via cell sorting based on Cripto-1 expression. We determined by FACS analysis that the proportion of the Cripto-1-positive melanoma cells in the aggressive melanoma cell line C8161 was approximately 2% (Figure 2A). These Cripto-1 sorted cells (C8161-CR-1) were much smaller and more spindle shaped than the parental cells and tended to form aggregates (Figure 2A). C8161-CR-1 cells also grew at a much slower rate than the parental cells, and Western blot analysis of cell lysates from these cells showed lower levels of Nodal and increased expression of the embryonic stem cell related transcription factors Oct4 and Nanog compared to parental C8161 cells used as the control (Figure 2B). The transcription factors Oct4 and Nanog, along with Cripto-1, have been shown to regulate pluripotentiality, self-renewal, cellular commitment and differentiation of mouse and human ES cells ^37–40. Moreover, Cripto-1 has been shown to be a direct target gene in mouse and human ES cells for Nanog and Oct4 ^{39, 40}. The Cripto-1-positive melanoma cells, however, lost their expression of Cripto-1, Nanog and Oct4 after subsequent passages. The experiment was repeated, and immediately after following FACS sorting for expression of Cripto-1, these melanoma cells were cultured in feeder-cell conditioned embryonic stem cell media (cESCM) rather than traditional complete RPMI cell culture media, and they formed spherical colonies and expressed higher levels of Cripto-1, Oct4, Nanog and MDR1 as compared to the control C8161 cells (Figure 3A). At passage 8, the sphere forming properties of these cells were still maintained as long as they continued to grow in cESCM. When seeded back in complete RPMI media, these cells no longer formed spheres and grew again in a monolayer similar to the parental cell line (Figure 3A).

Figure 2.

The aggressive melanoma cell line C8161 contains a small population of CR-1 positive cells (C8161/CR-1+). The result from the FACS analysis shown in panel A describes a small population (~ 2%) of CR-1-positive cells present in C8161. The sorted C8161/CR-1+ cells are relatively smaller and more spindle-shaped as compared to the parental C8161 cells. In panel B, Western blot analysis of cell lysates obtained from the C8161/CR-1+ cells shows increased expression of CR-1 and of the stem cell associated transcription factors, Oct4 and Nanog, as compared to lysates from parental C8161.

Figure 3.

When C8161/CR-1+ cells are cultured in conditioned embryonic stem cell medium (cESCM) they begin to form spherical colonies as shown in A (i) which are not formed by the parental C8161 cells grown in the same media and shown in A (ii). However, when C8161/CR-1+ cells that have been previously cultured in cESCM are returned to RPMI media, they lose the sphere forming property and grow in monolayer A (iii) similar to that of the C8161 parental cells. In panel B, Western blot analysis of cell lysates from C8161/CR-1+ cells cultured in cESCM continue to show increased expression of Cripto-1, Oct4 and Nanog and also show increased expression of MDR1, as compared to lysates from C8161 parental cells used as control.

Future perspectives

The highly plastic nature of human malignant melanoma, the expression of stem cell-related markers like Oct4 and MDR1, and activation of certain signaling molecules shown to play an important role during the maintenance of pluripotency and self renewal in hESCs, such as Nodal, support the hypothesis that a stem cell-like population, possibly MSC, may be present in melanoma. The identification of MSC may help elucidate the role that these cells play in determining the aggressive phenotype in melanoma. Methods of identifying and targeting these cells could help reduce the well documented resistance to current chemotherapeutic agents and may translate into clinically relevant management strategies for recurrence and metastasis. However, several important questions still need to be addressed. For instance, Nodal has been clearly demonstrated to represent an important signaling component for aggressive melanoma cells, although the role of the Nodal co-receptor, Cripto-1, in these cells is not clearly understood. Cripto-1 has been suggested to play an important role during tumorigenesis by promoting cell proliferation and survival, as well as other changes such as epithelial-to-mesenchymal transition ⁴¹. Although high levels of Cripto-1 have been detected in certain malignancies such as cancers of the breast, stomach and colon, as well as in uveal melanoma ⁴², in cutaneous melanoma Cripto-1 expression is restricted to only a small fraction of the population ¹². Future studies will determine whether this subpopulation of Cripto-1-positive cells is sufficient to sustain Nodal signaling in cutaneous melanoma. Recent studies, however, have reported that Nodal signaling can occur independently of Cripto-1, ⁴³ suggesting that Cripto-1 may not play a significant role during Nodal signaling in cutaneous melanoma. Nevertheless, the fact that Cripto-1 is a target gene of the embryonic stem cell transcription factors Nanog and Oct4, and that Cripto-1 has been implicated in the maintenance of self renewal and pluripotency in stem cells ^{39, 40}, suggest that Cripto-1 expression may be exploited to identify and isolate cells with potential stem cell-like characteristics in cancer. In fact, our preliminary findings indicate that the small fraction of Cripto-1 cells isolated from metastatic melanoma cells expresses Nanog and Oct4 and shows stem cell-like growth characteristics. Future studies involving clonogenic assays or results from serial in vivo transplantation will determine whether these cells actually represent a cancer stem cell population and ultimately demonstrate whether Cripto-1 can be used to identify and target MSC.

Abbreviations

MDR1

Multidrug Resistance Protein-1

RGP

Radial-Growth Phase

VGP

Vertical-Growth Phase

hESC

human Embryonic Stem Cell

MSC

Melanoma Stem Cells

TMEFF1

Tomoregulin

ABC

ATP-binding cassette

cESCM

conditioned Embryonic Stem Cell Media