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Intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance and a slow-cycling state in bone marrow-disseminated tumor cells.

Nakamura T, Shinriki S, Jono H, Guo J, Ueda M, Hayashi M, Yamashita S, Zijlstra A, Nakayama H, Hiraki A, Shinohara M, Ando Y - Oncotarget (2015)

Bottom Line: Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly.Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation.These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
Dormant or slow-cycling disseminated tumor cells (DTCs) in bone marrow (BM) are resistant to conventional therapy in various cancers including head and neck squamous cell carcinoma (HNSCC), although the molecular mechanisms remain largely unknown. This study aimed to identify the intrinsic molecular mechanisms underlying drug resistance in BM-DTCs. We used in vivo selection of the human HNSCC cell line HEp3, which mimics non-proliferative BM-DTCs in mice, to establish BM-DTC-derived (BM-HEp3) and lung metastases-derived (Lu-HEp3) sublines. Both sublines had higher migration activity and shortened survival in a murine xenograft model compared with parental (P-HEp3) cells. Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly. The drug resistance and slow-cycling state of BM-HEp3 cells depended on enhanced positive feedback of the signaling axis of stromal cell-derived factor-1 (SDF-1)-C-X-C chemokine receptor-4 (CXCR4) via their overexpression. Interestingly, BM-DTCs highly expressed transforming growth factor-beta 2 (TGF-β2) to maintain SDF-1-CXCR4 overexpression. Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation. These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.

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Autocrine SDF-1-CXCR4 signaling maintains a slow-cycling state and drug resistance in BM-derived DTCs(A) mRNA expression of CXCR4 (left) and SDF-1 (middle), and CXCR4 protein expression (right) in the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines, cultured in serum-free medium for 24 hours, was determined via qRT-PCR and Western blotting, respectively. *P < .001. n.s., not significant. (B) BM-HEp3 cells were treated with AMD3100 (5 μM) for 24 hours, after which SDF-1 mRNA expression was determined via qRT-PCR. †P < .05. (C) The sublines were treated with AMD3100 (5 μM) for 24 hours, after which cell numbers were counted. †P < .05. (D) Cells were treated with cisplatin (5 μg/ml) with or without AMD3100 (5 μM) for 48 hours, after which cell numbers were counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. †P < .05,§P < .01. n.s., not significant. Values are means ± SEM of triplicate samples.
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Figure 3: Autocrine SDF-1-CXCR4 signaling maintains a slow-cycling state and drug resistance in BM-derived DTCs(A) mRNA expression of CXCR4 (left) and SDF-1 (middle), and CXCR4 protein expression (right) in the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines, cultured in serum-free medium for 24 hours, was determined via qRT-PCR and Western blotting, respectively. *P < .001. n.s., not significant. (B) BM-HEp3 cells were treated with AMD3100 (5 μM) for 24 hours, after which SDF-1 mRNA expression was determined via qRT-PCR. †P < .05. (C) The sublines were treated with AMD3100 (5 μM) for 24 hours, after which cell numbers were counted. †P < .05. (D) Cells were treated with cisplatin (5 μg/ml) with or without AMD3100 (5 μM) for 48 hours, after which cell numbers were counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. †P < .05,§P < .01. n.s., not significant. Values are means ± SEM of triplicate samples.

Mentions: The signaling mediated by the chemokine SDF-1 (also called CXCL12) and its cognate receptor CXCR4 has a central role in BM homing and is also required for the quiescence and retention of hematopoietic stem cells in the BM [7]. In view of these known functions, we compared the expression of SDF-1 and CXCR4 in BM-HEp3 cells with that in the other cell lines. As Figure 3A illustrates, gene expression of both SDF-1 and CXCR4 in BM-HEp3 cells was much higher than that in the other cell lines. No significant difference in expression of these genes between P-HEp3 and Lu-HEp3 cells was observed. We confirmed increased CXCR4 protein level in BM-HEp3 cells compared to the other cell lines (Figure 3A). These data indicated that SDF-1-CXCR4 was constitutively enhanced in BM-DTCs. Treatment with AMD3100, a CXCR4-specific inhibitor [24], dramatically suppressed SDF-1 transcription in BM-HEp3 cells, which indicated that SDF-1 expression depended an enhanced CXCR4 downstream signal (Figure 3B). Together, these data suggest that BM-DTCs maintained a positive feedback SDF-1-CXCR4 signaling loop.


Intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance and a slow-cycling state in bone marrow-disseminated tumor cells.

Nakamura T, Shinriki S, Jono H, Guo J, Ueda M, Hayashi M, Yamashita S, Zijlstra A, Nakayama H, Hiraki A, Shinohara M, Ando Y - Oncotarget (2015)

Autocrine SDF-1-CXCR4 signaling maintains a slow-cycling state and drug resistance in BM-derived DTCs(A) mRNA expression of CXCR4 (left) and SDF-1 (middle), and CXCR4 protein expression (right) in the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines, cultured in serum-free medium for 24 hours, was determined via qRT-PCR and Western blotting, respectively. *P < .001. n.s., not significant. (B) BM-HEp3 cells were treated with AMD3100 (5 μM) for 24 hours, after which SDF-1 mRNA expression was determined via qRT-PCR. †P < .05. (C) The sublines were treated with AMD3100 (5 μM) for 24 hours, after which cell numbers were counted. †P < .05. (D) Cells were treated with cisplatin (5 μg/ml) with or without AMD3100 (5 μM) for 48 hours, after which cell numbers were counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. †P < .05,§P < .01. n.s., not significant. Values are means ± SEM of triplicate samples.
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Related In: Results  -  Collection

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Figure 3: Autocrine SDF-1-CXCR4 signaling maintains a slow-cycling state and drug resistance in BM-derived DTCs(A) mRNA expression of CXCR4 (left) and SDF-1 (middle), and CXCR4 protein expression (right) in the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines, cultured in serum-free medium for 24 hours, was determined via qRT-PCR and Western blotting, respectively. *P < .001. n.s., not significant. (B) BM-HEp3 cells were treated with AMD3100 (5 μM) for 24 hours, after which SDF-1 mRNA expression was determined via qRT-PCR. †P < .05. (C) The sublines were treated with AMD3100 (5 μM) for 24 hours, after which cell numbers were counted. †P < .05. (D) Cells were treated with cisplatin (5 μg/ml) with or without AMD3100 (5 μM) for 48 hours, after which cell numbers were counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. †P < .05,§P < .01. n.s., not significant. Values are means ± SEM of triplicate samples.
Mentions: The signaling mediated by the chemokine SDF-1 (also called CXCL12) and its cognate receptor CXCR4 has a central role in BM homing and is also required for the quiescence and retention of hematopoietic stem cells in the BM [7]. In view of these known functions, we compared the expression of SDF-1 and CXCR4 in BM-HEp3 cells with that in the other cell lines. As Figure 3A illustrates, gene expression of both SDF-1 and CXCR4 in BM-HEp3 cells was much higher than that in the other cell lines. No significant difference in expression of these genes between P-HEp3 and Lu-HEp3 cells was observed. We confirmed increased CXCR4 protein level in BM-HEp3 cells compared to the other cell lines (Figure 3A). These data indicated that SDF-1-CXCR4 was constitutively enhanced in BM-DTCs. Treatment with AMD3100, a CXCR4-specific inhibitor [24], dramatically suppressed SDF-1 transcription in BM-HEp3 cells, which indicated that SDF-1 expression depended an enhanced CXCR4 downstream signal (Figure 3B). Together, these data suggest that BM-DTCs maintained a positive feedback SDF-1-CXCR4 signaling loop.

Bottom Line: Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly.Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation.These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
Dormant or slow-cycling disseminated tumor cells (DTCs) in bone marrow (BM) are resistant to conventional therapy in various cancers including head and neck squamous cell carcinoma (HNSCC), although the molecular mechanisms remain largely unknown. This study aimed to identify the intrinsic molecular mechanisms underlying drug resistance in BM-DTCs. We used in vivo selection of the human HNSCC cell line HEp3, which mimics non-proliferative BM-DTCs in mice, to establish BM-DTC-derived (BM-HEp3) and lung metastases-derived (Lu-HEp3) sublines. Both sublines had higher migration activity and shortened survival in a murine xenograft model compared with parental (P-HEp3) cells. Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly. The drug resistance and slow-cycling state of BM-HEp3 cells depended on enhanced positive feedback of the signaling axis of stromal cell-derived factor-1 (SDF-1)-C-X-C chemokine receptor-4 (CXCR4) via their overexpression. Interestingly, BM-DTCs highly expressed transforming growth factor-beta 2 (TGF-β2) to maintain SDF-1-CXCR4 overexpression. Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation. These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.

Show MeSH
Related in: MedlinePlus