Abstract 1905: Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells
Authors: Ramirez-Fort Marigdalia K. , Liu He , Navarro Vicente , Meier Barbara , Levesque Mitch , Moy Jonathan , Vissicchio Jessica , Contassot Emmanuel , Kim Sae , Syed Talal , Zhang Michael , Tem Vincent , Christos Paul J. , Tagawa Scott T. , Bander Neil H. , Lange Christopher S. , French Lars E. 
Published: July 2017

Abstract
Background: Folate hydrolase-1 (FOLH1; NAALADase; PSMA) is a type II transmembrane protein that binds substrates with terminal glutamates. The MXXXL motif on the cytoplasmic N-terminal domain of FOLH1 interacts with clathrin and caveolin-1 to facilitate constitutive internalization upon substrate binding. J591, an established monoclonal antibody (AB) to FOLH1, is highly specific to and is effectively endocytosed after binding to the extracellular domain of FOLH1; J591 is presently being developed in the clinical trial setting, as a vehicle for AB-based brachytherapy in cancers that express FOLH1.
Physiological FOLH1 is expressed in cellular regions that are protected by tight-junctions or the blood-brain-barrier, and are therefore not targeted by circulating J591. Functionally, FOLH1 is responsible for cerebral glutamate production. In the oncological setting, FOLH1 is upregulated throughout prostate cancer cellular membranes, and is luminally expressed by cancer neovessels.
We have characterized neovascular FOLH1 expression in malignant melanoma (MM), a solid tumor of neural crest (NC) origin. Comparative RTqPCR analysis of normal skin, primary (p), and metastatic (m) MM revealed respective 10.64 and 18.21 -fold increases in FOLH1 gene-expression in pMM (p=0.0041) and mMM (p=0.042) samples as compared to normal skin. Immunohistochemistry of paraffin-embedded MM tumors revealed FOLH1 protein expression in the neovasculature of 4/11 (36%) of pMM and 9/14 (64%) of mMM cases; we noted a subset of keratinocytic and melanocytic FOLH1 positive cells.
These results, and the known functional role of glutamate production in a subset of NC-derived tissue, suggest that MM cells may also express cellular FOLH1.
Methods: Six (3 BRAF+; 3 BRAF-) MM cell lines were evaluated for cellular FOLH1 and Oct4 expression with J591-based and anti-Oct4 AB immunofluorescence (IF). Oct4 is a homeodomain binding protein encoded by POU5f1, widely accepted as a stem cell marker. FOLH1+ cell lines with permeablized and non-permeablized membranes were incubated with J591. The effect of γ-irradiation (0, 4.5, or 9 Gy) was also tested in FOLH1+ cell lines with harvesting 6 or 24 hours post-irradiation.
Results: Ten to 40% of MM cells demonstrated intracellular FOLH1 expression in 2 BRAF-/6 MM cell lines, with a predominant perinuclear and terminal dendritic distribution (e.g., axonal morphology). IF co-labeling with J591 (cytoplasmic) and antibodies to Oct4 (nuclear) identified possible stemness of FOLH1+ / Oct4+ MM cells. RT-PCR analysis of the irradiated cell lines demonstrated ~2-fold increased FOLH1 mRNA levels in 1/2 MM cell lines.
Conclusions: Herein is the first demonstration that MM cells express FOLH1. Given that FOLH1 glutamate production is closely linked to NC-originating glial cells, it can be reasonably postulated that this newly identified subset of FOLH1+/Oct4+ MM cells could be NC precursors, or cancer stem cells, for MM. Further, radiation-induced FOLH1 upregulation may increase the therapeutic ratio of AB-based brachytherapy.
Citation Format: Marigdalia K. Ramirez-Fort, He Liu, Vicente Navarro, Barbara Meier, Mitch Levesque, Jonathan Moy, Jessica Vissicchio, Emmanuel Contassot, Sae Kim, Talal Syed, Michael Zhang, Vincent Tem, Paul J. Christos, Scott T. Tagawa, Neil H. Bander, Christopher S. Lange, Lars E. French. Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1905. doi:10.1158/1538-7445.AM2017-1905