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. 2014 Aug 30;5(16):6854-66.
doi: 10.18632/oncotarget.2269.

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

Oihana Murillo-Sauca  1 , Man Ki Chung  2 , June Ho Shin  1 , Christina Karamboulas  3 , Shirley Kwok  4 , Young Ho Jung  2 , Richard Oakley  5 , James R Tysome  5 , Lovisa O Farnebo  2 , Michael J Kaplan  5 , Davud Sirjani  5 , Vasu Divi  6 , F Christopher Holsinger  6 , Chafeek Tomeh  5 , Anthony Nichols  7 , Quynh T Le  8 , A Dimitrios Colevas  9 , Christina S Kong  10 , Ravindra Uppaluri  11 , James S Lewis Jr  12 , Laurie E Ailles  3 , John B Sunwoo  2
Affiliations

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

Oihana Murillo-Sauca et al. Oncotarget. .

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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Conflict of interest statement

Conflict of Interest

The authors have no conflicts of interest with the studies presented here.

Figures

Figure 1
Figure 1. CD271 is heterogeneously expressed in SCCHN and marks the more tumorigenic population of cells
(A) Human primary SCCHN samples were stained with a monoclonal antibody against CD271 and assessed by immunohistochemistry and flow cytometry. The FACS plot is gated on the DAPI−lineage− population and co-stained for CD44 and CD271. (B) Two murine cell lines derived from oral tumors arising from DMBA-treated oral cavity mucosa were stained with a monoclonal antibody specific for murine CD271 and assessed by FACS. (C) Human cell lines derived from head and neck SCC were assessed for CD271 expression by FACS after staining with a monoclonal antibody to CD271.
Figure 2
Figure 2. CD271 loss-of-function results in decreased cell cycle progression in SCCHN
(A) Four cell lines derived from human SCCHN were assessed for in vitro growth kinetics by MTT assay and for in vivo tumor formation and growth by implantation into the flanks of Rag−/−γc−/− mice. (B) Knockdown of CD271 in human SCCHN cell lines by shRNA lentiviral constructs was assessed by FACS. (C) Effects of CD271 knockdown by shRNA expression on cell proliferation was assessed by MTT assay. In the cell lines with high expression of CD271 (UPCI:SCC-103 and PCI-13), there was a decrease in cell viability at the end of the MTT assay. Two different shRNAs were assessed: “sh3” = CD271 shRNA3. “sh5” = CD271 shRNA5. (D) The effects of the CD271 knockdown was reversed by co-expression of the CD271 coding sequence that lacked the 3′-UTR, which was targeted by the shRNA. In the FACS plots, the rescued expression of CD271 is demonstrated in the plot on the right. The bar graph represents the fold change in cell viability at the end of an MTT assay (relative to day 0). “c” = empty vector control or scramble RNA control. “p75”= CD271 coding sequence lacking the 3′-UTR. “sh3” = CD271 shRNA3. “sh5” = CD271 shRNA5 *p<0.01, **p<0.005. (E) PCI-13 cells were transduced with lentivirus expressing CD271 shRNA3, CD271 shRNA5, or a scrambled control RNA. Percentages of cells in the various phases of the cell cycle were assessed by FACS after staining with BrdU and PI; these percentages are quantified in the bar graph. *p<0.01.
Figure 3
Figure 3. CD271 loss-of-function results in decreased tumor initiation in vivo
(A) Schematic of in vivo competition assay. PCI-13 cells were transduced with either a lentivirus co-expressing shRNA targeting CD271 and mCherry or a lentivirus expressing Zsgreen. Cells were mixed 1:1 and injected into the flanks of Rag−/−γc−/− mice. Cell composition in tumors arising in these mice was assessed by FACS analysis of cells from dissociated tumors. (B) FACS analysis of input cells at time of implantation and cells from dissociated tumors at the termination of the experiment. The bar graphs depict the percentages of mCherry and Zsgreen expressing tumor cells from each cohort of recipient mice.
Figure 4
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.
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