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

Murillo-Sauca O, Chung MK, Shin JH, Karamboulas C, Kwok S, Jung YH, Oakley R, Tysome JR, Farnebo LO, Kaplan MJ, Sirjani D, Divi V, Holsinger FC, Tomeh C, Nichols A, Le QT, Colevas AD, Kong CS, Uppaluri R, Lewis JS, Ailles LE, Sunwoo JB - Oncotarget (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA. .

ABSTRACT
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.

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Related in: MedlinePlus

Targeting of CD271 with monoclonal antibody inhibits tumor formation and NGF-induced Erk phosphorylation(A) PCI-13 cells were incubated with either azide-free monoclonal antibody specific for CD271 or isotype control IgG for 30 min, washed, and then assessed for cell proliferation in vitro and tumor growth in vivo. The graph on the left shows MTT cell viability results over 8 days, expressed as fold-change from the day 0 baseline. The graph on the right shows tumor growth from cells injected subcutaneously into the flanks of Rag−/−γc−/− mice. Each cohort consists of 4 mice. Experiments were performed at least two times. (B) PCI-13 cells (grown in serum-free medium for 24 hrs) were incubated in vitro with monoclonal antibody to CD271 or isotype control IgG for 30 min and then with or without recombinant human NGF for 1 hr. Cell lysates were subjected to gel electrophoresis, and Western immunoblot analysis was performed with antibody specific for phosphorylated-Erk (p-Erk1/2), total Erk, and actin. All experiments performed two or more times.
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Figure 4: Targeting of CD271 with monoclonal antibody inhibits tumor formation and NGF-induced Erk phosphorylation(A) PCI-13 cells were incubated with either azide-free monoclonal antibody specific for CD271 or isotype control IgG for 30 min, washed, and then assessed for cell proliferation in vitro and tumor growth in vivo. The graph on the left shows MTT cell viability results over 8 days, expressed as fold-change from the day 0 baseline. The graph on the right shows tumor growth from cells injected subcutaneously into the flanks of Rag−/−γc−/− mice. Each cohort consists of 4 mice. Experiments were performed at least two times. (B) PCI-13 cells (grown in serum-free medium for 24 hrs) were incubated in vitro with monoclonal antibody to CD271 or isotype control IgG for 30 min and then with or without recombinant human NGF for 1 hr. Cell lysates were subjected to gel electrophoresis, and Western immunoblot analysis was performed with antibody specific for phosphorylated-Erk (p-Erk1/2), total Erk, and actin. All experiments performed two or more times.

Mentions: The inhibition of in vivo tumor formation with CD271 loss-of-function suggested that this molecule might be a viable therapeutic target in SCCHN. To address this hypothesis, we targeted the receptor with a monoclonal antibody specific for NGFR. Incubation of PCI-13 SCCHN cells with the antibody resulted in a significant reduction in cell proliferation in vitro compared to cells treated with isotype control IgG (Figure 4A). Furthermore, treatment of the cells with the anti-NGFR antibody resulted in significant reduction in tumor growth in vivo compared to isotype control – treated cells.


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

Murillo-Sauca O, Chung MK, Shin JH, Karamboulas C, Kwok S, Jung YH, Oakley R, Tysome JR, Farnebo LO, Kaplan MJ, Sirjani D, Divi V, Holsinger FC, Tomeh C, Nichols A, Le QT, Colevas AD, Kong CS, Uppaluri R, Lewis JS, Ailles LE, Sunwoo JB - Oncotarget (2014)

Targeting of CD271 with monoclonal antibody inhibits tumor formation and NGF-induced Erk phosphorylation(A) PCI-13 cells were incubated with either azide-free monoclonal antibody specific for CD271 or isotype control IgG for 30 min, washed, and then assessed for cell proliferation in vitro and tumor growth in vivo. The graph on the left shows MTT cell viability results over 8 days, expressed as fold-change from the day 0 baseline. The graph on the right shows tumor growth from cells injected subcutaneously into the flanks of Rag−/−γc−/− mice. Each cohort consists of 4 mice. Experiments were performed at least two times. (B) PCI-13 cells (grown in serum-free medium for 24 hrs) were incubated in vitro with monoclonal antibody to CD271 or isotype control IgG for 30 min and then with or without recombinant human NGF for 1 hr. Cell lysates were subjected to gel electrophoresis, and Western immunoblot analysis was performed with antibody specific for phosphorylated-Erk (p-Erk1/2), total Erk, and actin. All experiments performed two or more times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196168&req=5

Figure 4: Targeting of CD271 with monoclonal antibody inhibits tumor formation and NGF-induced Erk phosphorylation(A) PCI-13 cells were incubated with either azide-free monoclonal antibody specific for CD271 or isotype control IgG for 30 min, washed, and then assessed for cell proliferation in vitro and tumor growth in vivo. The graph on the left shows MTT cell viability results over 8 days, expressed as fold-change from the day 0 baseline. The graph on the right shows tumor growth from cells injected subcutaneously into the flanks of Rag−/−γc−/− mice. Each cohort consists of 4 mice. Experiments were performed at least two times. (B) PCI-13 cells (grown in serum-free medium for 24 hrs) were incubated in vitro with monoclonal antibody to CD271 or isotype control IgG for 30 min and then with or without recombinant human NGF for 1 hr. Cell lysates were subjected to gel electrophoresis, and Western immunoblot analysis was performed with antibody specific for phosphorylated-Erk (p-Erk1/2), total Erk, and actin. All experiments performed two or more times.
Mentions: The inhibition of in vivo tumor formation with CD271 loss-of-function suggested that this molecule might be a viable therapeutic target in SCCHN. To address this hypothesis, we targeted the receptor with a monoclonal antibody specific for NGFR. Incubation of PCI-13 SCCHN cells with the antibody resulted in a significant reduction in cell proliferation in vitro compared to cells treated with isotype control IgG (Figure 4A). Furthermore, treatment of the cells with the anti-NGFR antibody resulted in significant reduction in tumor growth in vivo compared to isotype control – treated cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA. .

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus