Latest Findings

Graft-versus-Host Disease-Related Cytokine-Driven Apoptosis Depends on p73 in Cytokeratin 15-Positive Target Cells.

Biol Blood Marrow Transplant. 2012 Jun;18(6):841-51. Epub 2012 Mar 30.

Zhan Q, Korngold R, Lezcano C, McKeon F, Murphy GF.

Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.

Acute graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation, involves cytotoxic soluble and cellular effectors that selectively induce apoptosis in normally apoptosis-resistant, cytokeratin 15 (K15)-expressing epithelial stem cells residing at the tips of rete ridges of human epidermis and in analogous rete-like prominences (RLPs) of murine dorsal lingual epithelium. The mechanisms whereby epithelial stem cells are rendered vulnerable to apoptosis during allostimulation are unknown. We hypothesized that GVHD-induced target cell injury may be related to pathways involving the p53 family that are constitutively expressed by epithelial stem cells and designed to trigger physiological apoptosis as a result of environmental danger signals. Among the p53 family members, we found that p73 protein and mRNA were preferentially expressed in K15(+) RLPs of murine lingual squamous epithelium. On in vitro exposure to recombinant TNF-α and IL-1 in an organ culture model previously shown to replicate early GVHD-like target cell injury, apoptosis was selectively induced in K15(+) stem cell regions and was associated with induction of phosphorylated p73, a marker for p73 activation, and apoptosis was abrogated in target tissue obtained from p73-deficient (p73(-/-)) mice. Evaluation of early in vivo lesions in experimental murine GVHD disclosed identical patterns of phosphorylated p73 expression that coincided with the onset of effector T cell infiltration and target cell apoptosis within K15(+) RLPs. This study is the first to suggest that paradoxical apoptosis in GVHD of physiologically protected K15(+) epithelial stem cells is explainable, at least in part, by cytokine-induced activation of suicide pathways designed to eliminate stem cells after exposure to deleterious factors perceived to be harmful to the host.

PMID: 22469882


SOX2 contributes to melanoma cell invasion.

Lab Invest. 2012 Mar;92(3):362-70. doi: 10.1038/labinvest.2011.188. Epub 2011 Dec 19.

Girouard SD, Laga AC, Mihm MC, Scolyer RA, Thompson JF, Zhan Q, Widlund HR, Lee CW, Murphy GF.

Program in Dermatopathology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.

The mechanisms of melanoma invasion are poorly understood despite extensive inquiry. SRY (sex determining region Y)-box 2 (SOX2) is an embryonic stem cell transcription factor that has recently been discovered to be expressed in human melanoma where it is associated with dermal invasion and primary tumor thickness. To assess the potential role of SOX2 expression in melanoma invasion, we examined patient melanomas and humanized melanoma xenografts, and noted preferential SOX2 expression in cells that interfaced and infiltrated dermal stroma. Experimental knockdown (KD) of SOX2 mRNA and protein in A2058 melanoma cells with high constitutive SOX2 expression resulted in 4.5-fold decreased invasiveness in vitro compared with controls (P<0.0001). Conversely, when G361 cells that normally express low SOX2 were transduced to overexpress SOX2 mRNA and protein, a 3.8-fold increase in invasiveness was observed (P=0.0004). Among 84 invasion-related genes, RT-PCR screening revealed that SOX2 KD resulted in striking decrease in matrix metalloproteinase-3 (MMP-3), an endopeptidase associated with cleavage of the extracellular matrix. Quantitatively, SOX2 KD diminished MMP-3 mRNA by 87.8%. MMP-3 KD in SOX2-expressing A2058 cells served to inhibit invasion, although to a lesser degree than SOX2 KD. Finally, immunostaining of patient and xenograft melanomas revealed coordinate SOX2 and MMP-3 expression in regions of stromal infiltration. These data implicate SOX2 expression in melanoma invasion, and suggest a role for MMP-3 as one potential mediator of this process.

PMID: 22184093


SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study.

Exp Dermatol. 2011 Apr;20(4):339-45. doi: 10.1111/j.1600-0625.2011.01247.x.

Laga AC, Zhan Q, Weishaupt C, Ma J, Frank MH, Murphy GF.

Department of Pathology, Program in Dermatopathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.

SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the peripheral nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma.

PMID: 21410764


VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth.

Cancer Res. 2011 Feb 15;71(4):1474-85. Epub 2011 Jan 6.

Frank NY, Schatton T, Kim S, Zhan Q, Wilson BJ, Ma J, Saab KR, Osherov V, Widlund HR, Gasser M, Waaga-Gasser AM, Kupper TS, Murphy GF, Frank MH.

Division of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

Melanoma growth is driven by malignant melanoma-initiating cells (MMIC) identified by expression of the ATP-binding cassette (ABC) member ABCB5. ABCB5(+) melanoma subpopulations have been shown to overexpress the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE1 and are associated with CD31(-) vasculogenic mimicry (VM), an established biomarker associated with increased patient mortality. Here we identify a critical role for VEGFR-1 signaling in ABCB5(+) MMIC-dependent VM and tumor growth. Global gene expression analyses, validated by mRNA and protein determinations, revealed preferential expression of VEGFR-1 on ABCB5(+) tumor cells purified from clinical melanomas and established melanoma lines. In vitro, VEGF induced the expression of CD144 in ABCB5(+) subpopulations that constitutively expressed VEGFR-1 but not in ABCB5(-) bulk populations that were predominantly VEGFR-1(-). In vivo, melanoma-specific shRNA-mediated knockdown of VEGFR-1 blocked the development of ABCB5(+) VM morphology and inhibited ABCB5(+) VM-associated production of the secreted melanoma mitogen laminin. Moreover, melanoma-specific VEGFR-1 knockdown markedly inhibited tumor growth (by > 90%). Our results show that VEGFR-1 function in MMIC regulates VM and associated laminin production and show that this function represents one mechanism through which MMICs promote tumor growth.

PMID: 21212411


Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells.

Wound Repair Regen. 2010 Jul-Aug;18(4):426-32. Epub 2010 Jun 8.

Song G, Nguyen DT, Pietramaggiori G, Scherer S, Chen B, Zhan Q, Ogawa R, Yannas IV, Wagers AJ, Orgill DP, Murphy GF.

Division of Plastic Surgery, Brigham & Women’s Hospital, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.

PMID: 20546556


Isolation of tumorigenic circulating melanoma cells.

Biochem Biophys Res Commun. 2010 Oct 23. [Epub ahead of print]

Ma J, Lin JY, Alloo A, Wilson BJ, Schatton T, Zhan Q, Murphy GF, Waaga-Gasser AM, Gasser M, Hodi FS, Frank NY, Frank MH.

Transplantation Research Center, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA;
Department of Dermatology, Brigham & Women’s Hospital, Boston, MA, USA.

Circulating tumor cells (CTC) have been identified in several human malignancies, including malignant melanoma. However, whether melanoma CTC are tumorigenic and cause metastatic progression is currently unknown. Here we isolate for the first time viable tumorigenic melanoma CTC and demonstrate that this cell population is capable of metastasis formation in human-to-mouse xenotransplantation experiments. The presence of CTC among peripheral blood mononuclear cells (PBMC) of murine recipients of subcutaneous (s.c.) human melanoma xenografts could be detected based on mRNA expression for human GAPDH and/or ATP-binding cassette subfamily B member 5 (ABCB5), a marker of malignant melanoma-initiating cells previously shown to be associated with metastatic disease progression in human patients. ABCB5 expression could also be detected in PBMC preparations from human stage IV melanoma patients but not healthy controls. The detection of melanoma CTC in human-to-mouse s.c. tumor xenotransplantation models correlated significantly with pulmonary metastasis formation. Moreover, prospectively isolated CTC from murine recipients of s.c. melanoma xenografts were capable of primary tumor initiation and caused metastasis formation upon xenotransplantation to secondary murine NOD-scid IL2Rγ(null) recipients. Our results provide initial evidence that melanoma CTC are tumorigenic and demonstrate that CTC are capable of causing metastatic tumor progression. These findings suggest a need for CTC eradication to inhibit metastatic progression and provide a rationale for assessment of therapeutic responses of this tumorigenic cell population to promising emerging melanoma treatment modalities.

PMID: 20977885


The embryonic morphogen, Nodal, is associated with channel-like structures in human malignant melanoma xenografts.

J Cutan Pathol. 2010 Apr;37 Suppl 1:19-25.

McAllister JC, Zhan Q, Weishaupt C, Hsu MY, Murphy GF.

Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA.

Formation of channel-like structures, also termed vasculogenic mimicry (VM), describes the ability of aggressive melanoma cells to form PAS-positive anastomosing structures that correlate with tumor virulence. This phenomenon may indicate differentiation plasticity, a feature melanoma cells may share with stem cells in the developing embryo. Recent studies have indicated that VM and tumorigenicity of human malignant melanoma may depend on the signaling pathways of an embryonic morphogen, Nodal. However, given the secretory nature of Nodal protein and melanoma cell heterogeneity, it remains unclear whether the Nodal-expressing cells participate directly or indirectly in VM that is potentially related to tumorigenic growth. We have developed a humanized murine xenograft model in which developing human melanomas may be sequentially studied during early stages of tumorigenic growth within a physiological human dermal microenvironment. Nodal protein localized diffusely to melanoma cell membranes, with occasional foci of accentuated reactivity in patterns suggestive of channel formation. Similar findings were detected in a limited number of patient-derived tumors. In situ hybridization confirmed Nodal mRNA to be restricted to tumor cells within xenografts that formed arborizing networks in patterns consistent with VM. These data indicate that Nodal gene expression is associated with formation of VM-like structures in a physiologically relevant model of human melanoma tumorigenesis, and further support a key role for Nodal expression in the formation of channel-like structures. The humanized xenograft model should be useful in future studies to define the mechanistic pathways responsible for VM and melanoma progression.

PMID: 20482672