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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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The SDF-1-CXCR4-dependent slow-cycling state and drug resistance in BM-DTCs requires TGF-β2(A) mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) cells was determined via qRT-PCR. *P < .05, †P < .01 compared with P-HEp3 cells unless otherwise indicated. (B) Expression of TGF-β2 (left), CXCR4 (middle), and SDF-1 (right) mRNA in P-HEp3 and BM-HEp3 cells was measured by means of qRT-PCR 48 hours after TGF-β2 siRNA transfection. *P < .05, §P < .005. n.s., not significant. (C) The numbers of P-HEp3, Lu-HEp3, and BM-HEp3 cells were determined 48 hours after transfection with control or TGF-β2 siRNA. *P < .05. (D) BM-HEp3 cells were treated with TGF-β1 (5 ng/ml) or TGF-β3 (5 ng/ml) for 48 hours, and then cell numbers were counted. *P < .05,†P < .01. (E) Cisplatin (5 μg/ml) was added to P-HEp3, Lu-HEp3, and BM-HEp3 cells at 48 hours after control or TGF-β2 siRNA transfection, followed by incubation for 48 hours in serum-free conditions. Cell numbers were then counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. *P < .05, ‡P < .001. Values are means ± SEM of triplicate samples.
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Figure 4: The SDF-1-CXCR4-dependent slow-cycling state and drug resistance in BM-DTCs requires TGF-β2(A) mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) cells was determined via qRT-PCR. *P < .05, †P < .01 compared with P-HEp3 cells unless otherwise indicated. (B) Expression of TGF-β2 (left), CXCR4 (middle), and SDF-1 (right) mRNA in P-HEp3 and BM-HEp3 cells was measured by means of qRT-PCR 48 hours after TGF-β2 siRNA transfection. *P < .05, §P < .005. n.s., not significant. (C) The numbers of P-HEp3, Lu-HEp3, and BM-HEp3 cells were determined 48 hours after transfection with control or TGF-β2 siRNA. *P < .05. (D) BM-HEp3 cells were treated with TGF-β1 (5 ng/ml) or TGF-β3 (5 ng/ml) for 48 hours, and then cell numbers were counted. *P < .05,†P < .01. (E) Cisplatin (5 μg/ml) was added to P-HEp3, Lu-HEp3, and BM-HEp3 cells at 48 hours after control or TGF-β2 siRNA transfection, followed by incubation for 48 hours in serum-free conditions. Cell numbers were then counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. *P < .05, ‡P < .001. Values are means ± SEM of triplicate samples.

Mentions: In view of the above findings, we next investigated the mechanisms of CXCR4 overexpression in BM-DTCs. In several types of cancer including HNSCC, TGF-β is a critical regulator of not only hematopoietic stem cell hibernation in the BM [25] but also of metastatic processes, including tumor cell colonization, cell dormancy, and metastatic progression, in distant organs such as bone [18,26-28]. We therefore first assessed expression of all TGF-β isoforms—TGF-β1, TGF-β2, and TGF-β3. Our real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed significantly increased TGF-β2 gene expression in BM-HEp3 cells compared with P-HEp3 cells (Figure 4A). In contrast, TGF-β2 expression in Lu-HEp3 cells was much lower than that in P-HEp3 cells. A notable result was that in BM-HEp3 cells, expression of TGF-β1 and TGF-β3 was significantly decreased compared with that in P-HEp3 cells, whereas Lu-HEp3 cells showed increased TGF-β1 expression compared with P-HEp3 cells.


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)

The SDF-1-CXCR4-dependent slow-cycling state and drug resistance in BM-DTCs requires TGF-β2(A) mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) cells was determined via qRT-PCR. *P < .05, †P < .01 compared with P-HEp3 cells unless otherwise indicated. (B) Expression of TGF-β2 (left), CXCR4 (middle), and SDF-1 (right) mRNA in P-HEp3 and BM-HEp3 cells was measured by means of qRT-PCR 48 hours after TGF-β2 siRNA transfection. *P < .05, §P < .005. n.s., not significant. (C) The numbers of P-HEp3, Lu-HEp3, and BM-HEp3 cells were determined 48 hours after transfection with control or TGF-β2 siRNA. *P < .05. (D) BM-HEp3 cells were treated with TGF-β1 (5 ng/ml) or TGF-β3 (5 ng/ml) for 48 hours, and then cell numbers were counted. *P < .05,†P < .01. (E) Cisplatin (5 μg/ml) was added to P-HEp3, Lu-HEp3, and BM-HEp3 cells at 48 hours after control or TGF-β2 siRNA transfection, followed by incubation for 48 hours in serum-free conditions. Cell numbers were then counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. *P < .05, ‡P < .001. Values are means ± SEM of triplicate samples.
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Figure 4: The SDF-1-CXCR4-dependent slow-cycling state and drug resistance in BM-DTCs requires TGF-β2(A) mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) cells was determined via qRT-PCR. *P < .05, †P < .01 compared with P-HEp3 cells unless otherwise indicated. (B) Expression of TGF-β2 (left), CXCR4 (middle), and SDF-1 (right) mRNA in P-HEp3 and BM-HEp3 cells was measured by means of qRT-PCR 48 hours after TGF-β2 siRNA transfection. *P < .05, §P < .005. n.s., not significant. (C) The numbers of P-HEp3, Lu-HEp3, and BM-HEp3 cells were determined 48 hours after transfection with control or TGF-β2 siRNA. *P < .05. (D) BM-HEp3 cells were treated with TGF-β1 (5 ng/ml) or TGF-β3 (5 ng/ml) for 48 hours, and then cell numbers were counted. *P < .05,†P < .01. (E) Cisplatin (5 μg/ml) was added to P-HEp3, Lu-HEp3, and BM-HEp3 cells at 48 hours after control or TGF-β2 siRNA transfection, followed by incubation for 48 hours in serum-free conditions. Cell numbers were then counted. Results are expressed as a percentage relative to cells without cisplatin in each experimental group. *P < .05, ‡P < .001. Values are means ± SEM of triplicate samples.
Mentions: In view of the above findings, we next investigated the mechanisms of CXCR4 overexpression in BM-DTCs. In several types of cancer including HNSCC, TGF-β is a critical regulator of not only hematopoietic stem cell hibernation in the BM [25] but also of metastatic processes, including tumor cell colonization, cell dormancy, and metastatic progression, in distant organs such as bone [18,26-28]. We therefore first assessed expression of all TGF-β isoforms—TGF-β1, TGF-β2, and TGF-β3. Our real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed significantly increased TGF-β2 gene expression in BM-HEp3 cells compared with P-HEp3 cells (Figure 4A). In contrast, TGF-β2 expression in Lu-HEp3 cells was much lower than that in P-HEp3 cells. A notable result was that in BM-HEp3 cells, expression of TGF-β1 and TGF-β3 was significantly decreased compared with that in P-HEp3 cells, whereas Lu-HEp3 cells showed increased TGF-β1 expression compared with P-HEp3 cells.

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