Limits...
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.

Show MeSH

Related in: MedlinePlus

Phenotypes of BM-derived DTCs(A) Schematic representation of the procedure used for in vivo selection (left panel). HEp3 cells expressing green fluorescent protein (GFP) (5 × 106) were injected subcutaneously (S.C.) into mice. At 30-40 days after injection, HEp3 cells in the primary site (P-HEp3) and DTCs in lung and BM were isolated and then expanded in monolayer culture. After subconfluent growth, lung- and BM-derived cells were injected subcutaneously into mice again. These transplantations were repeated five times, and the resultant sublines derived from BM and lung were called BM-HEp3 and Lu-HEp3, respectively. Phase-contrast and corresponding images merged with GFP fluorescence for P-HEp3, Lu-HEp3, and BM-HEp3 cells are shown (right panels). Scale bars indicate 400 μm. (B) Representative images of cell morphology of the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines. Scale bars indicate 50 μm. Arrows and arrowheads indicate filopodia-like and dendrite- or axon-like protrusions, respectively. (C) The HEp3 sublines were wounded by scratching and were then incubated in serum-free medium for 12 hours. Cell migration into the wound area was visualized with a phase-contrast microscope and photographed. Representative photographs are shown (left panels), and the quantitative results provide the means ± SEM of triplicate samples (right panel). *P < .01. (D) Tumor growth after the HEp3 sublines were injected subcutaneously into mice. The graph shows mean tumor growth rates ± SD for four animals per experimental condition. (E) Kaplan-Meier plots of overall survival of each experimental group. †P < .05 (log-rank test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359213&req=5

Figure 1: Phenotypes of BM-derived DTCs(A) Schematic representation of the procedure used for in vivo selection (left panel). HEp3 cells expressing green fluorescent protein (GFP) (5 × 106) were injected subcutaneously (S.C.) into mice. At 30-40 days after injection, HEp3 cells in the primary site (P-HEp3) and DTCs in lung and BM were isolated and then expanded in monolayer culture. After subconfluent growth, lung- and BM-derived cells were injected subcutaneously into mice again. These transplantations were repeated five times, and the resultant sublines derived from BM and lung were called BM-HEp3 and Lu-HEp3, respectively. Phase-contrast and corresponding images merged with GFP fluorescence for P-HEp3, Lu-HEp3, and BM-HEp3 cells are shown (right panels). Scale bars indicate 400 μm. (B) Representative images of cell morphology of the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines. Scale bars indicate 50 μm. Arrows and arrowheads indicate filopodia-like and dendrite- or axon-like protrusions, respectively. (C) The HEp3 sublines were wounded by scratching and were then incubated in serum-free medium for 12 hours. Cell migration into the wound area was visualized with a phase-contrast microscope and photographed. Representative photographs are shown (left panels), and the quantitative results provide the means ± SEM of triplicate samples (right panel). *P < .01. (D) Tumor growth after the HEp3 sublines were injected subcutaneously into mice. The graph shows mean tumor growth rates ± SD for four animals per experimental condition. (E) Kaplan-Meier plots of overall survival of each experimental group. †P < .05 (log-rank test).

Mentions: To clarify the mechanism underlying chemotherapeutic drug resistance in dormant or slow-cycling DTCs in the BM, we established BM- and lung-derived DTC sublines (Figure 1A). We injected HEp3 cells expressing green fluorescent protein subcutaneously into mice. After 4-5 weeks, we isolated HEp3 cells from the injection site, which we designated the parental line P-HEp3, and DTCs from the BM and the lung metastases. We expanded these two groups of DTCs in culture and then reinjected them into mice. We repeated this in vivo transplantation five times. Isolated DTCs from the BM and the lung metastases after the fifth transplantation were named BM-HEp3 and Lu-HEp3, respectively (Figure 1A, left panel). GFP expression of P-HEp3, Lu-HEp3, and BM-HEp3 cells was confirmed (Figure 1A, right panels). Consistent with previous reports [1,18,20], although overt metastases were observed in the lung at 5 weeks at the latest after injection, visible skeletal metastases did not occur throughout the five transplantations (data not shown). We analyzed the phenotypic characteristics of these BM- and lung-derived sublines and compared them with those of P-HEp3.


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)

Phenotypes of BM-derived DTCs(A) Schematic representation of the procedure used for in vivo selection (left panel). HEp3 cells expressing green fluorescent protein (GFP) (5 × 106) were injected subcutaneously (S.C.) into mice. At 30-40 days after injection, HEp3 cells in the primary site (P-HEp3) and DTCs in lung and BM were isolated and then expanded in monolayer culture. After subconfluent growth, lung- and BM-derived cells were injected subcutaneously into mice again. These transplantations were repeated five times, and the resultant sublines derived from BM and lung were called BM-HEp3 and Lu-HEp3, respectively. Phase-contrast and corresponding images merged with GFP fluorescence for P-HEp3, Lu-HEp3, and BM-HEp3 cells are shown (right panels). Scale bars indicate 400 μm. (B) Representative images of cell morphology of the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines. Scale bars indicate 50 μm. Arrows and arrowheads indicate filopodia-like and dendrite- or axon-like protrusions, respectively. (C) The HEp3 sublines were wounded by scratching and were then incubated in serum-free medium for 12 hours. Cell migration into the wound area was visualized with a phase-contrast microscope and photographed. Representative photographs are shown (left panels), and the quantitative results provide the means ± SEM of triplicate samples (right panel). *P < .01. (D) Tumor growth after the HEp3 sublines were injected subcutaneously into mice. The graph shows mean tumor growth rates ± SD for four animals per experimental condition. (E) Kaplan-Meier plots of overall survival of each experimental group. †P < .05 (log-rank test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359213&req=5

Figure 1: Phenotypes of BM-derived DTCs(A) Schematic representation of the procedure used for in vivo selection (left panel). HEp3 cells expressing green fluorescent protein (GFP) (5 × 106) were injected subcutaneously (S.C.) into mice. At 30-40 days after injection, HEp3 cells in the primary site (P-HEp3) and DTCs in lung and BM were isolated and then expanded in monolayer culture. After subconfluent growth, lung- and BM-derived cells were injected subcutaneously into mice again. These transplantations were repeated five times, and the resultant sublines derived from BM and lung were called BM-HEp3 and Lu-HEp3, respectively. Phase-contrast and corresponding images merged with GFP fluorescence for P-HEp3, Lu-HEp3, and BM-HEp3 cells are shown (right panels). Scale bars indicate 400 μm. (B) Representative images of cell morphology of the P-HEp3 (P), Lu-HEp3 (Lu), and BM-HEp3 (BM) sublines. Scale bars indicate 50 μm. Arrows and arrowheads indicate filopodia-like and dendrite- or axon-like protrusions, respectively. (C) The HEp3 sublines were wounded by scratching and were then incubated in serum-free medium for 12 hours. Cell migration into the wound area was visualized with a phase-contrast microscope and photographed. Representative photographs are shown (left panels), and the quantitative results provide the means ± SEM of triplicate samples (right panel). *P < .01. (D) Tumor growth after the HEp3 sublines were injected subcutaneously into mice. The graph shows mean tumor growth rates ± SD for four animals per experimental condition. (E) Kaplan-Meier plots of overall survival of each experimental group. †P < .05 (log-rank test).
Mentions: To clarify the mechanism underlying chemotherapeutic drug resistance in dormant or slow-cycling DTCs in the BM, we established BM- and lung-derived DTC sublines (Figure 1A). We injected HEp3 cells expressing green fluorescent protein subcutaneously into mice. After 4-5 weeks, we isolated HEp3 cells from the injection site, which we designated the parental line P-HEp3, and DTCs from the BM and the lung metastases. We expanded these two groups of DTCs in culture and then reinjected them into mice. We repeated this in vivo transplantation five times. Isolated DTCs from the BM and the lung metastases after the fifth transplantation were named BM-HEp3 and Lu-HEp3, respectively (Figure 1A, left panel). GFP expression of P-HEp3, Lu-HEp3, and BM-HEp3 cells was confirmed (Figure 1A, right panels). Consistent with previous reports [1,18,20], although overt metastases were observed in the lung at 5 weeks at the latest after injection, visible skeletal metastases did not occur throughout the five transplantations (data not shown). We analyzed the phenotypic characteristics of these BM- and lung-derived sublines and compared them with those of P-HEp3.

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