Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more persistent strategies