Cisplatin induces Bmi-1 and enhances the stem cell fraction in head and neck cancer

Abstract

Recent evidence has unveiled a subpopulation of highly tumorigenic, multipotent cells capable of self-renewal in head and neck squamous cell carcinomas (HNSCCs). These unique cells, named here cancer stem cells (CSCs), proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that CSCs are found in perivascular niches and rely on endothelial cell-secreted factors [particularly interleukin-6 (IL-6)] for their survival and self-renewal in HNSCC. Here, we hypothesized that cisplatin enhances the stem cell fraction in HNSCC. To address this hypothesis, we generated xenograft HNSCC tumors with University of Michigan-squamous cell carcinoma 22B (UM-SCC-22B) cells and observed that cisplatin treatment increased (P = .0013) the fraction of CSCs [i.e., aldehyde dehydrogenase activity high and cluster of differentiation 44 high (ALDH(high)CD44(high))]. Cisplatin promoted self-renewal and survival of CSCs in vitro, as seen by an increase in the number of orospheres in ultralow attachment plates and induction in B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and octamer-binding transcription factor 4 expression. Cisplatin-resistant cells expressed more Bmi-1 than cisplatin-sensitive cells. IL-6 potentiated cisplatin-induced orosphere formation generated when primary human HNSCC cells were sorted for ALDH(high)CD44(high) immediately after surgery and plated onto ultralow attachment plates. IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in UM-SCC-22B cells, whereas IL-6-induced extracellular signal-regulated kinase (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Notably, cisplatin-induced Bmi-1 was inhibited by interleukin-6 receptor blockade in parental and cisplatin-resistant cells. Taken together, these results demonstrate that cisplatin enhances the fraction of CSCs and suggest a mechanism for resistance to cisplatin therapy in head and neck cancer.

Figure 1

Cisplatin enhances the fraction of CSCs in HNSCC. (A) Schematic drawing depicts the experimental design. Xenograft tumors were generated by the transplantation of human HNSCC cells (UM-SCC-22B) and primary human endothelial cells (HDMEC) in SCID mice. When tumors reached an average size of 200 mm³, mice began to receive weekly intraperitoneal injections (5 mg/kg cisplatin or vehicle). (B) Photomicrographs of histologic sections immunostained for CD44 and Bmi-1 from xenograft tumors treated with cisplatin or vehicle controls. (C) Graph depicts the fraction of CSCs (as defined by ALDH^highCD44^high) in xenograft tumors treated with cisplatin or vehicle control (n = 11). Asterisk depicts P = .0013. (D and E) Graphs depict the viability of UM-SCC-22B (D) and UM-SCC-1 (E) cells, as determined by the SRB assay. Cells were preincubated for 1 hour with 20 ng/ml rhIL-6 and then coincubated with cisplatin (0–20 µM) and rhIL-6 for 24 to 72 hours. (F and G) Flow cytometric analysis of ALDH/CD44 status of UM-SCC-22B cells treated with rhIL-6 and/or cisplatin. Cells were preincubated for 1 hour with rhIL-6 and then coincubated with cisplatin (0 or 2 µM) and rhIL-6 (0–50 ng/ml) for 24 hours.

Figure 2

Cisplatin and IL-6 enhance Bmi-1 and the stemness of HNSCC cells. (A and B) Primary human HNSCC cells were sorted for ALDH and CD44 immediately after surgery. ALDH^highCD44^high cells were cultured in ultralow attachment plates for 10 days. (A) Photomicrographs depict orospheres (nonadherent spheres of > 25 HNSCC cells) after treatment with cisplatin (0–2 µM) and/or rhIL-6 (20 ng/ml) or vehicle control. (B) Graph depicts the number of orospheres generated with primary human HNSCC cells treated with cisplatin (0–2 µM) and/or rhIL-6 (0 or 20 ng/ml). Asterisk depicts P < .05. (C) Western blot analysis depicts the expression of Bmi-1 in UM-SCC-22A and UM-SCC-22B cells treated with cisplatin (0–2 µM) and/or rhIL-6 (0 or 20 ng/ml) for 24 hours. (D) Western blot analysis depicts the expression of CD44 in UM-SCC-22A and UM-SCC-22B cells treated with cisplatin (0–2 µM) and/or rhIL-6 (0 or 20 ng/ml) for 24 hours.

Figure 3

Effect of cisplatin on key signaling pathways involved in the regulation of stemness. HNSCC cells were serum starved overnight and then treated with cisplatin (0–5 µM), rhIL-6 (0–50 ng/ml), and/or anti-IL-6 neutralizing antibody (0–10 µg/ml) for 30 minutes. (A–C) Western blot analysis for phosphorylated and total STAT3, ERK, and AKT in UM-SCC-22B and UM-SCC-1 cells. (D) Western blot analysis for CDK4 in UM-SCC-22B and UM-SCC-1 cells.

Figure 4

Cisplatin-resistant cells display cancer stemlike features. Cisplatin-resistant cells were generated by subculturing these cells in presence of increasing concentrations of cisplatin from 0.5 to 12 µM during a 3-month period. (A) Photomicrographs of parental cells (UM-SCC-22B) and cells resistant to 1 µM cisplatin (UM-SCC-22BCis1), 6 µM cisplatin (UM-SCC-22BCis6), or 12 µM cisplatin (UM-SCC-22BCis12). (B) Graph depicts number of orospheres generated with parental UM-SCC-22B cells or cisplatin-resistant cells (UM-SCC-22BCis12) treated with cisplatin (0 or 2 µM) and/or rhIL-6 (0 or 20 ng/ml) for 10 days in ultralow attachment plates. Different lowercase letters depict P < .05. (C) Western blot analysis of the expression of Bmi-1 in cisplatin-resistant cells (UM-SCC-22BCis1, UM-SCC-22BCis4, UM-SCC-22BCis6, and UM-SCC-22BCis12), compared to parental cells (UM-SCC-22B). (D and E) Western blot analysis of the expression of Bmi-1 (D) or Oct-4 (E) in cisplatin-resistant cells (UM-SCC-22BCis1 and UM-SCC-22BCis12), compared to parental cells (UM-SCC-22B). Cells were treated with cisplatin (0–2 µM) and/or rhIL-6 (0–20 ng/ml) for 24 hours.

Figure 5

IL-6 activates STAT3 signaling and induces Bmi-1 expression in cisplatin-resistant cells. (A) Western blot analyses for phosphorylated and total STAT3 and ERK in cisplatin-resistant cells (UM-SCC-22BCis1, UM-SCC-22BCis4, UM-SCC-22BCis6, and UM-SCC-22BCis12), compared to parental cells (UM-SCC-22B). (B) Western blot analyses for expression of IL-6R in cisplatin-resistant cells (UM-SCC-22BCis series), compared to parental cells (UM-SCC-22B). Cells were treated with cisplatin (0–2 µM) and/or rhIL-6 (0 or 20 ng/ml) for 24 hours. (C) Western blot analysis of Bmi-1 in cisplatin-resistant cells (UM-SCC-22BCis1), compared to parental cells. Cells were treated with cisplatin (0–2 µM) and/or rhIL-6 (0 or 20 ng/ml) in presence of anti-IL-6 or anti-IL-6R antibody for 24 hours.