Reprogramming metastatic tumor cells with embryonic microenvironments: Targeting tumor cell plasticity
Authors: Mary Hendrix, Elisabeth Seftor, Richard Seftor, Naira Margaryan, Jolanta Topczewska, Brian Nickoloff, Jacek Topczewski, Jennifer Kasemeier-Kulesa, Paul Kulesa, Lynne-Marie Postovit
Journal: Cancer Research (2007)

SY10-01  Aggressive tumor cells share many characteristics in common with embryonic progenitors -- contributing to the conundrum of tumor cell plasticity. Recent studies using embryonic models of human stem cells, the zebrafish, and the chick have shown the reversion of the metastatic phenotype of aggressive melanoma cells, and revealed the convergence of embryonic and tumorigenic signaling pathways, which may help to identify new targets for therapeutic intervention. Key to developing novel therapeutic strategies is understanding the molecular mechanism(s) involved in reprogramming the phenotype of multipotent tumor cells. This presentation will summarize three embryonic models used to reverse the metastatic melanoma phenotype, and highlight the prominent signaling pathways that have emerged as noteworthy targets for future consideration.  The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells requires special attention as we contemplate new therapeutic strategies targeting the elusive metastatic phenotype. Furthermore, both normal stem cells and tumor cells are profoundly influenced by the bi-directional communication with their respective microenvironments, which dictates cell fate determination and behavior. Although the concept of relating cancer to stem cells has recently gained popularity, primarily due to the identification of specific tumor stem cells, there is a great deal to learn about their regulation -- resulting in normal or aberrant behavior.  Scientific evidence in support of these early concepts, which now appear to be remarkably insightful, would derive from several early studies in the 1970’s and 1980’s. Following the general theme that an embryonic microenvironment might have the capacity to reverse the metastatic phenotype of cancer cells, Mintz and Illmensee demonstrated that a mouse embryonic blastocyst microenvironment could suppress the tumorigenic phenotype of teratocarcinoma cells, and the developmental plasticity of the tumor cells was manifested as they contributed to the formation of normal tissue. Later, Pierce and colleagues proposed that an embryonic microenvironment capable of differentiating a stem cell lineage should be able to reprogram cancers derived from that lineage. Indeed support for this concept demonstrating the tumor-inhibiting properties of embryonic microenvironments was derived from the testing of a variety of cancer cell lines. One example by Gerschenson and colleagues from these studies showed the inability of B16 murine melanoma cells to form tumors following exposure to microenvironmental factors derived from the embryonic skin of a developing mouse. Additional experiments (performed by Bissell and colleagues) documented a similar tumor-suppressing microenvironment in a chick model -- where Rous sarcoma virus failed to induce sarcomas in the embryos.  Technological advances in microscopy and molecular biology have allowed scientists to extend observations made by the pioneers working at the intersection of developmental biology and cancer biology. Recent investigations have revealed unique insights into the molecular reprogramming of metastatic melanoma cells exposed to the embryonic microenvironments of human embryonic stem cells (hESCs), the zebrafish, and the chick embryo. These observations have led to the discovery of a novel signaling pathway in melanoma that underlies stem cell plasticity and provides a new target for therapeutic intervention.  Global gene analyses have revealed the unexpected finding that aggressive melanoma tumor cells express genes (and proteins) associated with a variety of cell types (including progenitor cells), while simultaneously down-regulating genes specific to their parental melanocytic lineage. These intriguing findings support the premise that aggressive melanoma cells acquire a multipotent, plastic phenotype -- a concept that challenges our current thinking of how to target tumor cells with stem cell-like properties. Indeed, a major undertaking to identifying the molecular pathways regulating stem cell plasticity is examining the potential epigenetic role of the microenvironment on the emergence of cell phenotype.  Based on the similarities associated with the plastic phenotype of embryonic stem cells and multipotent melanoma tumor cells, together with the observation that human embryonic stem cells (hESCs) do not form tumors during blastocyst implantation -- although they do form teratomas when transplanted into immunodeficient mice, one must assume that the microenvironment exerts dominant control over stem cell fate. Therefore, we hypothesized that the microenvironment of hESCs might possess the potential to reprogram the metastatic melanoma phenotype. To explore this possibility, we developed a model that allowed hESCs to “condition” a 3D matrix, which would subsequently receive multipotent metastatic melanoma cells. This experimental strategy demonstrated that the hESC microenvironment can dramatically alter the behavior of metastatic melanoma cells. Specifically, exposure of the tumor cells to the 3D matrices preconditioned by the hESCs induced melanoma cells to form spheroids similar to the colonies formed by hESCs. Interestingly, the conditioned media from hESCs did not alter the phenotype of the melanoma cells, indicating that hESCs can influence the tumor cell phenotype via modification of the immediate microenvironment. Biochemical and molecular analyses of the melanoma cells (which were amelanotic) exposed to the hESC microenvironment revealed the epigenetic induction of a melanocyte-specific phenotype marker, Melan-A. In contrast, a normal melanocyte microenvironment does not share the ability of the hESC microenvironment to epigenetically reprogram metastatic melanoma cells. Changes in the biological behavior of melanoma cells exposed to the microenvironment of hESCs were manifested by significant reductions in the invasive capacity of the tumor cells in vitro and in tumorigenic potential in vivo, suggesting suppressive cues associated with the hESC microenvironment.  The embryonic neural crest microenvironment is the second model system to explore tumor cell reprogramming and metastatic ability. The neural crest (NC) is a multipotent cell population that emerges from the neural tube and invades the embryo in a programmed manner to form peripheral neurons and glia and the entire facial and visceral skeleton of the vertebrate head. A subpopulation of NC cells distribute throughout the embryo and differentiate into pigment cells (from which melanocytes and melanoma derive) to protect the body from UV radiation. Based on the migratory and invasive properties shared by both NC cells and multipotent melanoma tumor cells, we utilized an embryonic chick model to specifically explore the possibility of reverting melanoma cells toward their NC cellular derivatives. When GFP-labeled human metastatic melanoma cells were transplanted into the chick embryonic microenvironment, the melanoma cells invaded host NC targets, did not form tumors, and a subset of these tumor cells were reprogrammed to a NC cell-like phenotype. Specifically, the transplanted melanoma cells populated host peripheral structures in a programmed manner, including the branchial arches, dorsal root and sympathetic ganglia. A subpopulation of melanoma cells that invaded the chick periphery was reprogrammed to express the melanocyte-associated protein Melan-A and the neuronal marker Tuj1. These intriguing results demonstrate that metastatic melanoma cells can respond to developmental cues and suggest that factors unique to the NC embryonic milieu may be implemented to reprogram melanoma cells to a more benign melanocytic cell type. Thus, improved insights into the mechanisms that govern the tumor cell reprogramming, studied within the chick neural crest microenvironment, might therefore be relevant for the identification of new cancer therapies.  To further our study into the convergence of embryonic and tumorigenic signaling pathways contributing to plasticity, we utilized the developing zebrafish as a biosensor for tumor derived signals. The data demonstrate that aggressive melanoma cells, but not poorly aggressive cells, can direct the fate of pluripotent zebrafish stem cells. Specifically, the aggressive tumor cells were able to orchestrate the formation of ectopic embryonic axes by secreting Nodal, a potent embryonic morphogen. Highlighting the potential role of this molecule in melanoma dissemination, Nodal protein expression was positively correlated with melanoma metastasis and inhibition of Nodal signaling reduced melanoma cell invasiveness. Moreover, depletion of Nodal signaling in aggressive melanoma cells caused the reversion of these cells toward a more melanocytic phenotype, and resulted in the abrogation of tumorigenicity. From these data, we uncovered a key role for Nodal signaling in tumor cell plasticity and tumorigenicity, thereby providing a novel molecular target for regulating tumor progression.