Abstract
BackgroundBone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC). Intracellular BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) during cellular development. However, little is known about the role or regulation of BMP signaling in HNSCC CSCs.MethodsTwo CSC-like populations, CD44high/BMI1high and CD44high/ALDHhigh, were enriched from HNSCC cell lines and evaluated for the expression of SMURF1 by qRT-PCR, flow cytometry, and immunoblotting. The activation status of BMP signaling in these populations was determined by using immunoblotting to detect phosphorylated SMAD1/5/8 (pSMAD1/5/8) levels. Knockdown of SMURF1 transcripts by RNA interference was used to assess the role of SMURF1 in BMP signaling and CSC maintenance. Loss of CSC-like phenotypes following SMURF1 knockdown was determined by changes in CD44high levels, cellular differentiation, and reduction in colony formation.ResultsPopulations of enriched CSC-like cells displayed decreased levels of pSMAD1/5/8 and BMP signaling target gene ID1 while SMURF1, CD44, and BMI1 were highly expressed when compared to non-CSC populations. Stable knockdown of SMURF1 expression in CSC-like cells increased pSMAD1/5/8 protein levels, indicating the reactivation of BMP signaling pathways. Decreased expression of SMURF1 also promoted adipogenic differentiation and reduced colony formation in a three-dimensional culture assay, indicating loss of tumorigenic capacity. The role of SMURF1 and inhibition of BMP signaling in maintaining a CSC-like population was confirmed by the loss of a CD44high expressing subpopulation in SMURF1 knockdown cells.ConclusionsOur findings suggest that inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs, and that this inhibition is mediated by SMURF1. Targeting SMURF1 and restoring BMP signaling may offer a new therapeutic approach to promote differentiation and reduction of CSC populations leading to reduced drug resistance and disease recurrence.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-260) contains supplementary material, which is available to authorized users.
Highlights
Bone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC)
CSC-like characteristics To study the role of BMP signaling in modulating CSC populations, we established stable sphere cultures to enrich for CSC-like cells from three HNSCC cell lines, TR146, SCC-58, and UMSCC-17B
Because CD44 and BMI1 expression were consistently expressed in sphere cells and sphere-derived cells (SDCs) from the three cell lines, sphere cells grown under nonadherent conditions for 10 to 14 days were used as representative CSC-like populations, and all SDCs were derived from these populations for the remaining studies
Summary
Bone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC). Little is known about the role or regulation of BMP signaling in HNSCC CSCs. Methods: Two CSC-like populations, CD44high/BMI1high and CD44high/ALDHhigh, were enriched from HNSCC cell lines and evaluated for the expression of SMURF1 by qRT-PCR, flow cytometry, and immunoblotting. Evidence suggests that a subpopulation of cells, known as cancer stem cells (CSCs), possess the potential for self-renewal, multipotent differentiation, and tumorigenesis [5,7,8], and these cells may contribute to the aggression and dismal prognosis of HNSCC. ALDH activity correlates strongly with increased resistance to chemo- and radiotherapy [15,16], and the combination of ALDH activity with CD44 expression is more selective for CSC-like populations than either marker alone [17,18,19,20]
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