对“失巢凋亡”（anoikis）的抵抗作用在肿瘤扩散和转移中可能扮演一个重要角色。“失巢凋亡”是一种形式的细胞程序死亡，是由与细胞外基质和其他细胞脱离接触而诱发的。来自荷兰癌症研究所的一个小组，从认为“失巢凋亡”抑制是癌症细胞在循环过程中生存的一个先决条件这个观点出发，想弄清对这一性质进行筛选是否可作为在整个基因组范围内对转移基因的一种功能筛选方法。这种筛选方法在大鼠的小肠上皮细胞中发现了作为“失巢凋亡”一种强力抑制剂的神经营养受体 TrkB，表达TrkB的细胞的确易发生癌变，能够转移。这一发现也许可解释过度表达 TrkB的人类肿瘤为什么具有侵略性。
Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB
Metastasis is a major factor in the malignancy of cancers, and is often responsible for the failure of cancer treatment. Anoikis (apoptosis resulting from loss of cell–matrix interactions) has been suggested to act as a physiological barrier to metastasis; resistance to anoikis may allow survival of cancer cells during systemic circulation, thereby facilitating secondary tumour formation in distant organs1-3. In an attempt to identify metastasis-associated oncogenes, we designed an unbiased, genome-wide functional screen solely on the basis of anoikis suppression. Here, we report the identification of TrkB, a neurotrophic tyrosine kinase receptor4, 5, as a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells. By activating the phosphatidylinositol-3-OH kinase/protein kinase B pathway, TrkB induced the formation of large cellular aggregates that survive and proliferate in suspension. In mice, these cells formed rapidly growing tumours that infiltrated lymphatics and blood vessels to colonize distant organs. Consistent with the ability of TrkB to suppress anoikis, metastases—whether small vessel infiltrates or large tumour nodules—contained very few apoptotic cells. These observations demonstrate the potent oncogenic effects of TrkB and uncover a specific pro-survival function that may contribute to its metastatic capacity, providing a possible explanation for the aggressive nature of human tumours that overexpress TrkB.
Figure 1 Functional cloning and validation of TrkB as a suppressor of anoikis. a, Parental or RasV12-expressing RIE cells seeded into ULC tissue culture plates and photographed at 50 magnification after 3 days (top). The bottom panel shows subsequent transfer of the cells to adhesive tissue culture plates, followed by fixation and crystal violet staining 2 days later. b, Outline of the screen. See Methods for details. c, Suppression of anoikis by clone ME-3, relative to a GFP-control, photographed at 50 (top) and 1 (bottom) magnification and at indicated time points. d, RIE cells expressing cDNAs as indicated, seeded on adhesive plates and photographed at 200 magnification. e, RIE cells expressing cDNAs as indicated, seeded into ULC plates and photographed in time at 50 (day 3) and 1 (day 14) magnification. f, Single cell suspensions of RIE cells expressing cDNAs as indicated, seeded into soft-agar and photographed at 50 magnification 1 week later.
Figure 2 Specific suppression of anoikis-associated apoptosis by TrkB. a, Control or TrkB-expressing RIE cells seeded into ULC plates and analysed for apoptosis over time by DiOC6(3) staining for FACS analysis30. Right- and left-hand peaks reflect living and apoptotic cells respectively. b, Immunoblotting for cleaved caspase-3 of lysates from RIE cells expressing indicated cDNAs, on adhesive (left) or ULC (right) plates, for 2 days. CDK4 serves as loading control. c, RIE cells expressing indicated cDNAs, seeded either in the presence (red) or absence (blue) of serum into ULC plates and analysed for apoptosis 3 days later, as in a. Results with 0.1% serum were similar to those with 0% (not shown). d, e, Rat1 fibroblasts co-expressing c-Myc and indicated cDNAs, on adhesive dishes, in the absence of serum for 3 days and analysed by FACS (d) and photographed at 200 magnification (e). Empty vector-transduced cells in 10% serum (Vector 10%) serve as control. f, Immunoblotting for serine 473-phosphorylated PKB (P-PKB) and total PKB of lysates from RIE cells expressing indicated cDNAs, on adhesive (left) or ULC plates (right) for 2 days. g, TrkB/BDNF co-expressing cells, either in the presence or absence of activated PKB, treated after spheroid formation with LY294002 (20 µM) or solvent and photographed 12 days later at 1 magnification. h, i, TrkB/BDNF-expressing cells treated after spheroid formation with rapamycin (20 nM), photographed at 50 magnification 2 days later (h) and subsequently used for immunoblotting for p70S6K and CDK4 as loading control (i). Similar results were obtained for 1-week treatment (not shown).
Figure 3 Tumorigenic potential of TrkB. a–c, Mice injected intravenously with RIE cells co-expressing a luciferase gene and indicated cDNAs, and subjected to in vivo imaging at indicated time points, with relative light units per pixel indicated in colour (a, c), or represented in a Kaplan–Meier survival plot (b). Note the relatively short latency of TrkB/BDNF-induced tumours and different imaging sensitivities used (c).
Figure 4 Metastatic potential of TrkB. a–j, Pathological characteristics of tumour processes after tail vein injection into nude mice. a, Large tumour nodule within liver. b, Clusters of tumour cells located in liver sinusoids and a tumour cell located in vena hepatica branch. c, Tumour nodule within liver, invading a vena portae branch. d, Large tumour within kidney. e, Detail of a renal tumour growing within lumen of large vein. f, Another plane of the section shown in e, demonstrating renal tumour extending into vein, activating the blood coagulation system and causing fibrin deposition (F). g, Detail of tumour in d, showing tumour cells located between renal tubules, and high proliferative index, as evident from nuclear MCM7 immunoreactivity. h, Representative photomicrograph of section in g, showing lack of apoptosis, as evident from absence of cleaved caspase-3 immunoreactivity. i, Tumour nodule (with edge of large tumour mass at bottom side of microphotograph), spreading into branch of coronary vein of the myocardium and showing high proliferative index, as evident from MCM7 immunopositivity. j, Representative photomicrograph of adjacent section of i, showing lack of apoptosis of tumour cells infiltrating coronary vein, as evidenced by a lack of expression of cleaved caspase-3. k, Splenic lymphoma from an unrelated mouse, showing numerous apoptotic cells, serving as positive control for cleaved caspase-3-specific immunohistochemistry. l–p, Pathological characteristics of tumour processes upon subcutaneous injection. Representative microphotographs showing the metastatic process in one mouse. l, Subcutaneously injected tumour cells infiltrating muscle tissue (arrows). m, Clusters of tumour cells infiltrating lymphatic vessels, at a distance from the subcutaneous tumour. n, Tumour infiltrating axillary lymph node (L) draining the tumour site. o, Large metastases at pleural side of the lungs (apical lobe of the right lung was removed to allow a better view of the tumour nodules). p, One of the metastases shown in o. Immunohistochemistry was performed for TrkB (a–e, m, n), MCM7 (g, i), or cleaved caspase-3 (h, j, k). f, l, p, Haematoxylin and eosin stainings. T and V mark tumours and vessels, respectively. Scale bars: 500 µm (a, p); 200 µm (c, e); 100 µm (f, k, l, n); 50 µm (b, g–j); 20 µm (m); 1 mm (d); 5 mm (o). Rows indicate four individual sets of photographs.
