CD271 Confers an Invasive and Metastatic Phenotype of Head and Neck Squamous Cell Carcinoma through the Upregulation of Slug.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is comprised of heterogeneous populations of cells, and CD271 (NGFR; p75NTR) has been associated with a tumor-initiating cell subpopulation. This study assessed the role of CD271 in modulating metastatic behavior in HNSCC.Experimental Design: CD271 was overexpressed in murine and human oral squamous cell carcinoma cells to assess the impact of CD271 activation on the invasive and metastatic phenotype of these cells, using in vitro and orthotopic in vivo modeling. Treatment with human nerve growth factor (NGF) to activate CD271, as well as shRNA knockdown of the CD271-upregulated Snai2 expression, was used to assess the mechanism of the CD271-induced invasive phenotype. Relevance of CD271 expression in human HNSCC was evaluated in patient-derived xenografts (PDX) and primary human oral cancers, annotated with clinical behavior characteristics and survival data.Results: Forced expression of CD271 resulted in a more invasive and metastatic phenotype. Slug, an epithelial-to-mesenchymal transition (EMT)-related transcription factor, encoded by Snai2, was highly expressed in MOC2-CD271 and HSC3-CD271, compared with respective parental cells. CD271 activation by NGF conferred enhanced invasiveness in CD271-overexpressing cells, which was abrogated by Snai2 knockdown. In PDXs and primary human HNSCC, CD271 expression correlated with higher Snai2 expression, greater nodal metastasis, and shorter disease-free survival.Conclusions: Activation of CD271 results in upregulation of Snai2/Slug, which, in turn, results in a more invasive phenotype and an enhanced capacity for metastasis to regional lymph nodes. These findings point to CD271 as a promising, therapeutic target for oral cancer metastasis. Clin Cancer Res; 24(3); 674-83. ©2017 AACR.

CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma.

Tumor-initiating cells (TICs) in squamous cell carcinoma of the head and neck (SCCHN) are best characterized by their surface expression of CD44. Although there is great interest in identifying strategies to target this population, no marker of these cells has been found to be functionally active. Here, we examined the expression of the purported marker of normal human oral epithelial stem cells, CD271. We show that CD271 expression is restricted to a subset of the CD44+ cells. Using xenograft assays, we show that the CD44+CD271+ subpopulation contains the most tumorigenic cells. Loss of CD271 function results in a block in the G2-M phase of the cell cycle and a profound negative impact on the capacity of these cells to initiate tumor formation in vivo. Incubation with recombinant NGF results in enhanced phosphorylation of Erk, providing additional evidence that CD271 is functionally active. Finally, incubation of SCCHN cells with antibody to CD271 results in decreased Erk phosphorylation and decreased tumor formation in vivo. Thus, our data are the first to demonstrate that CD271 more specifically identifies the TIC subpopulation within the CD44+ compartment in SCCHN and that this receptor is a functionally active and targetable molecule.

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.

CD44(+) cells have cancer stem cell-like properties in nasopharyngeal carcinoma.

BACKGROUND: A subpopulation of cells within a tumor appears to have the exclusive ability to initiate tumors, self-renew, and differentiate. These "cancer stem cells" (CSCs) are CD44(+) in several epithelial malignancies. We examined the potential of CD44 to identify the CSC population in nasopharyngeal carcinoma (NPC). METHODS: C666, an Epstein-Barr virus-positive (EBV(+) ) human NPC cell line, was stained for CD44 and sorted by fluorescence-activated cell sorting (FACS). CD44(+) and CD44(-) subpopulations were evaluated for (1) proliferative potential, (2) ability to differentiate, (3) expression of markers of epithelial-to-mesenchymal transition (EMT) and EBV genes, and (4) the ability to initiate tumors in vivo. Immunocompromised mice were injected with CD44(+) and CD44(-) populations to assess the tumor-initiating capacity. Immunohistochemistry for CD44 was performed on an 87-patient tissue microarray (TMA), and clinical correlations were examined. RESULTS: Heterogeneous expression of CD44 was seen among C666 cells. CD44(+) cells differentiated into CD44(-) cells, indicating a hierarchical relationship. Further, CD44(+) cells exhibited a more robust tumor-initiating capacity in the xenograft model. However, no differences were seen in proliferation rates in vitro, EBV gene expression, or expression of EMT markers between CD44(+) and CD44(-) subsets. Patient tumors were heterogeneous for CD44 staining, and a trend toward an association between CD44 expression and clinical outcome was observed. CONCLUSION: NPC contains a CD44(+) subpopulation with features consistent with CSCs. There was a trend toward an association between CD44 expression within NPC tumors and decreased time to local failure/relapse in patients.

ERK1/2 regulation of CD44 modulates oral cancer aggressiveness.

Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) incurs significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, we generated cell line models from 7,12-dimethylbenz(a)anthracene-induced murine primary OSCC capable of tumor formation upon transplantation into immunocompetent wild-type mice. Whereas several cell lines grew rapidly and were capable of metastasis, some grew slowly and did not metastasize. Aggressively growing cell lines displayed ERK1/2 activation, which stimulated expression of CD44, a marker associated with epithelial to mesenchymal transition and putative cancer stem cells. MEK (MAP/ERK kinase) inhibition upstream of ERK1/2 decreased CD44 expression and promoter activity and reduced cell migration and invasion. Conversely, MEK1 activation enhanced CD44 expression and promoter activity, whereas CD44 attenuation reduced in vitro migration and in vivo tumor formation. Extending these findings to freshly resected human OSCC, we confirmed a strict relationship between ERK1/2 phosphorylation and CD44 expression. In summary, our findings identify CD44 as a critical target of ERK1/2 in promoting tumor aggressiveness and offer a preclinical proof-of-concept to target this pathway as a strategy to treat head and neck cancer.