Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines
© Murai et al.; licensee BioMed Central Ltd. 2012
Received: 10 April 2012
Accepted: 18 July 2012
Published: 29 July 2012
Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines.
Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1) at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines.
Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both in vivo and in vitro.
Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells (ECs). Canine HSAs easily metastasize to other organs, and the mean survival time is less than 6 months even with surgical and chemotherapeutic interventions . Human angiosarcomas are also aggressive tumors that show a propensity for distant metastasis . Angiosarcomas occur rarely in humans, and no effective treatments have yet been developed. Because HSAs occur more commonly in dogs than in humans , it may be easier to study the progression of these tumors in dogs and to establish effective treatments that may also be applicable for human angiosarcomas.
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), along with their receptors, are overexpressed in human angiosarcomas and canine HSAs [2, 3]. These growth factors usually activate receptor tyrosine kinases (RTKs), which in turn activate downstream signaling pathways. Among these signaling pathways, MAPK/Erk and phosphatidyl-inositol-3 kinase /Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) are the major oncogenic signaling pathways [4, 5]. The MAPK/Erk pathway has been reported to be highly upregulated in benign endothelial tumors rather than in malignant tumors [6, 7]. In contrast, the PI3K/Akt pathway is known to be one of the important pathways in the manifestation of endothelial pathologies. For example, activated or mutated PI3K/Akt causes the development of HSA in chickens . Mutation of PTEN, a PI3K antagonist, has been reported in canine HSAs  and human angiosarcomas . Moreover, the Akt/mTOR pathway is upregulated in sporadic angiosarcomas in humans . However, the role of the PI3K/Akt/mTOR pathway has not been investigated in canine HSAs.
mTOR, a serine/threonine kinase, is highly conserved among animal species and regulates cell growth and cell cycle progression by controlling cap-dependent translation [12, 13]. mTOR exists as 2 distinct multi-protein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1, consisting of mTOR, raptor, and mLST8 (also known as GβL), is located downstream of PI3K/Akt and is activated by Akt via phophorylation at Ser2448. mTORC1 in turn phosphorylates the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and S6 kinase (S6K) . In its hypophosphorylated state, 4E-BP1 binds to and inhibits the activity of eIF4E, and 4E-BP1 phosphorylation induces the release of 4E-BP1 from eIF4E, which leads to subsequent mRNA translation . eIF4E is known to selectively stimulate several malignancy-related transcripts, including cyclin D1, bFGF, and VEGF , which are involved in growth, survival, and angiogenesis and are known to be overexpressed in human angiosarcomas [2, 17] and canine HSAs [3, 18]. mTORC2, consisting of mTOR, rictor, and mLST8, is located upstream of Akt and phosphorylates Akt at Ser473. Although RTK signaling is known to activate mTORC2 through the PI3K/PTEN pathway, less is known about mTORC2 signaling compared with that for mTORC1 [12, 20].
Because of the limited availability of human angiosarcoma [21, 22] or canine HSA [23, 24] cell lines, it was difficult to study deregulated signaling pathways in these tumors. We recently established xenograft canine HSA tumors from nude mice  and, in the present study, we present 7 canine HSA cell lines derived from the xenograft tumors. By using these established cell lines, we characterized the biological behavior of the cells in response to growth factors and disruption of signaling pathways. The primary aim of these studies is the identification of novel molecular targets for the treatment of canine HSAs.
To establish canine HSA cell lines, we used 3 xenograft canine HSA tumors, which were established from 3 spontaneous canine HSAs as described previously . Briefly, the xenograft tumor Ju was established from HSA tissue in the liver of a 10-year-old Labrador Retriever, Re was established from HSA tissue in the right atrium of a 10-year-old Golden Retriever, and Ud was established from HSA tissue in the spleen of an 11-year-old Papillion. These tumor tissues were subcutaneously transplanted into the right and left dorsal area of the trunk of 3-week-old male KSN/Slc nude mice (Japan SLC, Inc., Hamamatsu, Japan), and xenograft models were established after >5 passages. The xenografted tumor tissues were minced and sequentially digested in 0.1% collagenase Type I (Gibco, CA, USA) at 37°C for 15 min, and then 0.25% trypsin-EDTA (Gibco) at 37°C for 15 min. The cell suspension was subsequently filtered through a 70-μm cell strainer (BD Biosciences, NJ, USA), and then resuspended in Medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS, AusGeneX, Oxenford, Australia). The cells were cultured in a humidified incubator at 37°C with 5% CO2. Subconfluent cells were passaged after detachment with 0.25% trypsin-EDTA, and cell lines were established after >60 passages. For cloning, one cell per well was plated in separate 96-well plates (Thermo Scientific, MA, USA).
For measuring the growth curve and population doublings, the established cell lines were plated in 24-well plates (Thermo Scientific) at 5000 cells/well in 1 mL of Medium 199 containing 10% FBS. The cells were trypsinized and counted with a hemocytometer using trypan blue every 24 h. Triplicate wells were used for counting each cell line.
To examine the uptake of the acetylated low density lipoprotein (Ac-LDL) in HSA cell lines, subconfluent cells were incubated with 10 μg/mL DiI-Ac-LDL (Biomedical Technologies Inc., MA, USA) at 37°C for 4 h in Medium 199 according to the manufacturer’s instructions. After washing, the cells were observed with an inverted fluorescent microscope (Biozero BZ-8000, KEYENCE, Osaka, Japan) with a rhodamine filter. Human umbilical vein endothelial cells (HUVECs, Cell Applications Inc., CA, USA) were purchased and used as a positive control.
For measuring growth factors in cell supernatant, HSA cell lines were cultured under standard conditions in Medium 199 containing 10% FBS. After incubation for 72 h, the plates were washed with Hanks’ Balanced Salt Solution (HBSS, Sigma-Aldrich, MO, USA), and the medium was changed to Medium 199 containing 1% FBS. After further incubation for 24 h, the supernatant was stored at −80°C. The cells were trypsinized and counted with a hemocytometer using trypan blue. VEGF-A and bFGF concentrations in cell supernatant were determined using commercial ELISA kits for humans (Quantikine, R&D Systems, MN, USA) according to the manufacturer’s instructions since these kits were previously shown to have cross-reactivity with canine growth factors [26, 27].
Canine HSA cell lines were cultured to subconfluence under standard conditions in Medium 199 containing 10% FBS and were used for protein expression for VEGF-A and bFGF. After washing with phosphate-buffered saline without Ca2+ or Mg2+ [PBS (−)], the cells were incubated with Protein Block Serum-Free (Dako, Kyoto, Japan) for 30 min at room temperature (RT). The cells were incubated overnight at 4°C with primary antibodies for VEGF-A (mouse monoclonal antibody clone C-1, 1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and bFGF (rabbit polyclonal antibody, 1:200; Santa Cruz Biotechnology Inc.). The specific protein signals were visualized using the 3,3′-diaminobenzidinetetrahydrochloride (Liquid DAB + Substrate Chromogen System, Dako). The cells were counter-stained with Mayer’s hematoxylin.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
PCR primers and conditions
Annealing temperature (°C)
product size (bp)
249, 231, and 177
Cell proliferation assays
Cell proliferation assays were performed as previously described . Briefly, the established cell lines were plated at 1 × 103 cells per well in 200 μL Medium 199 containing 10% FBS in 96-well plates for 24 h. The cells were washed with HBSS, and the medium was replaced with Medium 199 containing 1% FBS. After 24 h of serum starvation, the cells were mixed with 0, 1, 10, 50, or 100 ng/mL of growth factor in Medium 199 containing 1% FBS or were changed to Medium 199 containing 10% FBS. Growth factors included recombinant human VEGF, bFGF, IGF-I, HGF, EGF, or PDGF-BB (R&D Systems), and all of these have been reported to induce cell growth in canine HSA cell lines except VEGF and PDGF-BB . Recombinant canine VEGF and HGF (R&D Systems) were also used. After 72-h incubation with growth factor or FBS, the relative viable cell number was assessed with the WST-1 assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Each experiment was repeated three times. Canine aortic endothelial cells (CnAOECs, Cell Applications Inc.) were purchased and used to examine the cell growth of normal canine ECs.
Canine HSA cell lines were cultured to 70–80% confluence under standard conditions in Medium 199 containing 10% FBS. Cells were then washed with HBSS and the medium was replaced with Medium 199 containing 1% FBS. After serum starvation for 24 h, the medium was replaced with Medium 199 containing 1% FBS or Medium 199 containing 10% FBS for 30 min. For PTEN expression, subconfluent cells grown in Medium 199 containing 10% FBS were used. After washing with PBS (−), the cells were lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology Inc.) with Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich). The concentrations of whole cell lysates were determined by modified Lowry method using the DC protein assay kit (Bio Rad, CA, USA). Equal amounts of protein (10 μg) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions on 10% polyacrylamide gels. After separation by SDS-PAGE, the proteins were transferred onto a PVFD-membrane (Millipore, MA, USA). Membranes were blocked with 2% ECL Blocking Agent (GE Healthcare Life Sciences, Buckinghamshire, UK) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at RT. The membranes were then incubated overnight at 4°C with primary antibodies for phosphorylated Akt (p-Akt Ser473, rabbit monoclonal antibody clone D9E, 1:1000, and Thr308, rabbit polyclonal antibody, 1:500; Cell Signaling Technology, MA, USA), Akt (rabbit polyclonal antibody, 1:1000; Cell Signaling Technology), p-p44/42 Erk1/2 (Thr202/Tyr204, rabbit polyclonal antibody, 1:1000; Cell Signaling Technology), p44/42 Erk1/2 (rabbit monoclonal antibody clone 137F5, 1:1000; Cell Signaling Technology), p-mTOR (Ser2448, rabbit monoclonal antibody clone D9C2, 1:1000; Cell Signaling Technology), mTOR (rabbit monoclonal antibody clone 7C10, 1:1000; Cell Signaling Technology), p-p70S6K (Thr389, mouse monoclonal antibody clone 1A5, 1:1000; Cell Signaling Technology), p70S6K (rabbit monoclonal antibody clone 49D7, 1:1000; Cell Signaling Technology), p-4E-BP1 (Thr37/46, rabbit monoclonal antibody clone 236B4, Thr70, rabbit polyclonal antibody, and Ser65, rabbit polyclonal antibody, all 1:1000; Cell Signaling Technology), 4E-BP1 (rabbit monoclonal antibody clone 53H11, 1:4000; Cell Signaling Technology), and PTEN (mouse monoclonal antibody clone A2B1, 1:200; Santa Cruz Biotechnology, Inc.). β-actin (mouse monoclonal antibody clone AC-15, 1:3000; Sigma-Aldrich) was used as a loading control. The specific protein signals were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Plus Western Blotting Detection System (GE Healthcare). CnAOECs were used to examine the protein expression for normal canine ECs.
Inoculation of cells and immunohistochemical staining
The established cell lines were harvested during logarithmic growth and prepared for injection in mice. Before injection, cells were trypsinized, counted, and washed twice with sterile PBS (−). A total of 1 × 106 cells were suspended in 0.2 ml of PBS (−) and injected subcutaneously into the right and left dorsal area of the trunk of 3-week-old male KSN/Slc mice. Five mice were used for each cell line. The mice were observed for tumor development twice a week, and the size of the resulting tumor was measured. After 9 weeks, or when the tumors grew to 10 mm in diameter, the mice were humanely sacrificed, and the tumors were immediately removed. If a detectable tumor was not formed in the mice within 30 days, the mice were sacrificed at this time. The removed tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) or used for immunohistochemical staining. Immunohistochemical staining was performed for CD31 (prediluted mouse monoclonal antibody clone JC70A; Dako), von Willebrand factor (vWF, prediluted rabbit polyclonal antibody; Dako), Ki-67 antigen (mouse monoclonal antibody clone MIB-1, 1:25; Dako), p-Akt (Ser473, rabbit polyclonal antibody, rabbit monoclonal antibody clone D9E, 1:50, Thr308, rabbit polyclonal antibody, 1:50; Cell Signaling Technology), and p-4E-BP1 (Thr37/46, rabbit monoclonal antibody clone 236B4, 1:1600; Cell Signaling Technology) on all tumors formed from the cell injections. The experiments were performed according to the guidelines for the care and use of laboratory animals and approved by the Committee for Animal Research and Welfare of Gifu University (No. 08015).
Student’s t test was used to determine statistical significance of the differences between the control and experimental data for the cell proliferation assay. Differences were considered statistically significant at p value of <0.05.
Morphology and growth of canine HSA cell lines
Expression of growth factor and growth factor receptor
One cell line (KDM/Re12) had a VEGF-A concentration of 201 pg/106 cells for 24 h in the cell supernatantas measured by ELISA, but bFGF was not found in the supernatant of any cell line. Immunocytochemical investigations for VEGF-A and bFGF revealed weak to moderate expression of these proteins observed in the cytoplasm of the cell lines (Figure 1B), in which the mRNA expression was found in RT-PCR.
Effects of growth factors on cell proliferation
Effects of serum stimulation on the MAPK/Erk and AKT/mTOR pathways
Deletion or mutation of PTEN is reported in some types of tumors, including vascular tumors [9, 10, 29], which causes constitutive activation of the PI3K/Akt pathway. PTEN protein was detected in all cell lines. The expression levels of PTEN in the KDM/JuA1 and KDM/JuB4 cells were lower than those in other cell lines and were not related to the phosphorylation levels of Akt (Figure 5B).
Tumor formation in nude mice
Tumor growth after subcutaneous injection of 1 × 10 6 cells of each canine HSA cell line
Growth (No. of animals)
Growth (No. of tumors)
Mean volume (mm3)
All tumors that formed were examined further for expression of the Akt/4E-BP1 pathway. Moderate to intense degrees of phosphorylation of Akt at Ser473was observed in both the nuclei and cytoplasm in all tumors (Figure 6H–K). On the other hand, weak to moderate phosphorylation of Akt at Thr308 was observed in both the nuclei and cytoplasm (Figure 6L–N), and this phosphorylation was not detected in tumors formed from Re21 injections (Figure 6O). 4E-BP1 at Thr37/46was highly phosphorylated in both the nuclei and cytoplasm in all tumors (Figure 6P–S).
We established 7 canine HSA cell lines from nude mice-xenograft canine HSAs . Although all original canine HSA xenograft tumors expressed mRNA for bFGF , some sub-lines derived from the same xenograft tumor lacked expression of bFGF. The differences in expression between xenograft tumors and subsequently derived sub-lines suggested that each xenograft tumor might contain a variety of tumor cells with different phenotypes. Each cell line had characteristics of ECs, which was confirmed by expression of CD31 mRNA and incorporation of DiI-Ac-LDL. However, vWF mRNA was not detected in any of the cell lines. The loss of vWF has also been reported in human angiosarcomas and canine HSA cell lines [22, 23] and occurs in undifferentiated malignant ECs . Therefore, the expression of vWF is of limited value for identifying malignant ECs , and CD31 is the most reliable EC marker [32, 33]. Unlike the expression levels in the cultured cell lines, expression of vWF and CD31 was observed in the tumors that formed after cell injections. vWF is produced by ECs and megakaryocytes, and adhere to collagen in the subendothelium . Tumors that formed after cell injection contained not only tumor cells but diverse cells, including red blood cells, inflammatory cells, and stromal cells. These cellular constituents may account for the differences in vWF expression observed between cultured cell lines and the resulting tumors after injection with these cells, but the exact cause of the differences remains unclear.
The established canine HSA cell lines expressed differing levels of mRNA for a variety of growth factors and their receptors. Although receptors were expressed in most of the cell lines, cell proliferation was stimulated only by the associated growth factors in the case of KDM/JuB4, in which proliferation was also stimulated by serum. Stimulated proliferation of 3 cell lines was observed in the presence of serum alone. A previous study with a canine HSA cell line showed that proliferation was stimulated by serum and the same growth factors that we used except for human VEGF and PDGF-BB . The previous study had a limitation, in that it analyzed only a single cell line. Because the present cell lines expressed both growth factors and their receptors, the lack of response to the growth factors may be the result of saturation of the receptors by growth factors in an autocrine or paracrine manner. Our findings suggest that serum may be a potent stimulator of cell proliferation in diverse types of canine HSA cells. In the serum, interleukins (ILs) such as IL-1α and IL-8 may be the primary stimulator since they are known to stimulate cell growth in canine HSAs as well as in normal ECs [35, 36]. However, a limitation of this study is that we could not evaluate the protein expression of receptors. Another possibility is that the lack of protein expression of the receptors may lead to unstimulated proliferation regardless of the mRNA expression.
In the present study, VEGF was detected in culture supernatant only in one cell line, even though mRNA and protein for VEGF was detected in all cell lines, and bFGF was not detected in the supernatant of any cell lines, including two cell lines that expressed mRNA and protein for bFGF. VEGF is known to regulate normal angiogenesis [37, 38] and is overexpressed in vascular tumors of both humans and dogs [2, 3]. In the previously reported canine HSA cell lines, VEGF [23, 24] and a small amount of bFGF  were detected using the same ELISA kit as that used in the present study. However, another study found that even though VEGF was present at high levels in the cytoplasm of activated ECs, it could not be detected in culture supernatant due to low levels of extracellular release . Because VEGF and bFGF mRNA and protein were expressed in the present cell lines but not in the supernatant, these growth factors are most likely to be contained only in the cytoplasm and were not released into the cell supernatant. It is also unknown whether these growth factors are released into the extracellular matrix in spontaneously occurring canine HSAs, in which both VEGF and bFGF are overexpressed .
The phosphorylation of Akt at Ser473 was not affected by FBS stimulation in all cell lines except KDM/Re12. In addition, the phosphorylation of mTORC1 at Ser2448 and 4E-BP1 at all residues was unchanged in all cell lines. In normal canine ECs, the phosphorylation of Akt at Ser473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 was increased in the presence of FBS, but not phosphorylation of 4E-BP1 at Thr37/46 or Thr70. 4E-BP1 is known to be sequentially phosphorylated on three residues: phosphorylation of Thr37/46 is followed by Thr70 and then Ser65. The phosphorylation of Thr37/46 is relatively unaffected by serum , whereas phosphorylation of Thr70 and Ser65 are stimulated by serum . However, a recent study indicated that different cell types as well as different stimuli lead to different 4E-BP1 phosphorylation . Furthermore, Ser65 of 4E-BP1 is an essential site for the control of translation initiation by release of 4E-BP1 from eIF4E . Our results suggest that phosphorylation of 4E-BP1 at Ser65 was the only site that was regulated in a serum-dependent manner in normal canine ECs, rather than Thr37/46 and Thr70. This indicates that Ser65 of 4E-BP1, Ser473 of Akt, and Ser2448 of mTORC1 were constitutively activated in the present cell lines. mTORC1 and mTORC2 are located both upstream and downstream of Akt, and Ser473 of Akt is directly phosphorylated by mTORC2 , whereas mTORC1 at Ser2448 is phosphorylated by Akt . The present findings suggest that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in a serum-independent manner, and was considered to be deregulated in the present cell lines compared with that in normal ECs. Consistent with the present results, constitutive phosphorylation of both Akt at Ser473 and 4E-BP1 is reported in lymphomas  and acute myeloid leukemia . Since these constitutively activated pathways are highly sensitive to molecular targeted therapies , the mTORC2/Akt/4E-BP1 pathway may be a novel target for treatment of canine HSAs. However, there is still possibility that mTORC1 and 4E-BP1 are phosphorylated independently of mTORC2, because mTORC1 was unaffected by serum regardless of increased phosphorylation of Akt at Ser473 in KDM/Re12. Another possibility is that phosphorylation of 4E-BP1 may not be caused by Akt nor mTORC1 because 4E-BP1 is known to be phosphorylated by p44/42 Erk1/2 . This is most likely to occur in KDM/Ud2 and KDM/Ud6 because the phosphorylation of Erk1/2 was unchanged in the presence of FBS.
Although 4E-BP1 was constitutively activated independent of FBS, cell proliferation was stimulated by serum in 4 cell lines. This stimulation seemed to be related to increased phosphorylation of p44/42 Erk1/2 Thr202/Tyr204, similar to that of normal canine ECs. The MAPK/Erk pathway regulates cell proliferation differently from the PI3K/Akt pathway [4, 5] and is not activated in human angiosarcomas . In contrast, the mTORC2/Akt/4E-BP1 pathway may regulate serum-independent cell proliferation because HSA cells could grow in serum-starved conditions. Another possibility is that constitutive mTORC2/Akt/4E-BP1 activation may lead to other effects besides cell proliferation since mTOR also regulates the cell cycle and anti-apoptosis [12, 13]. In KDM/Ud2 and KDM/Ud6, both the MAPK/Erk and mTORC2/Akt/4E-BP1 pathways were constitutively phosphorylated, and FBS stimulation failed to stimulate cell proliferation. RTKs are well-known activators of the MAPK/Erk and Akt/mTOR pathways, and mutations of RTKs in cancer lead to constitutive activation of these pathways [4, 5]. Therefore, the present constitutive activation of these two pathways may be result from aberrant activation of RTKs.
As opposed to phosphorylation of Akt at Ser473, the phosphorylation of Akt at Thr308 was affected by FBS stimulation and seemed to be correlated with the phosphorylation of p70S6K. Akt is usually phosphorylated at Thr308 by 3- phosphoinositide-dependent kinase, whereas Ser473 is phosphorylated by mTORC2 . Although both p70S6K and 4E-BP1 are located downstream of mTORC1 , recent studies have indicated that these 2 proteins are regulated by distinct signaling pathways in some types of cells. In normal ECs, p70S6K is regulated by mTORC1, and 4E-BP1 is regulated by Akt independently of the mTORC1 pathway . The mTORC1-independent regulation of 4E-BP1 has been also demonstrated in hematopoietic malignancies [29, 41]. Taken together, the phosphorylation of p70S6K and 4E-BP1 in the present cell lines was probably regulated by 2 different signaling pathways.
Deletion or mutation of PTEN is known to cause constitutive activation of the PI3K/Akt pathway in some types of tumors, including vascular tumors [9, 10, 29]. Deletion or point mutations have been reported in the C-terminal domain of PTEN in canine HSA cell lines . The antibody used in the present study also recognizes the C-terminal domain of PTEN. We found no evidence for deletion of PTEN in the present cell lines, despite constitutive phosphorylation of Akt at Ser473. It is known that constitutive activation of Akt is not always associated with the deletion or mutation of PTEN [9, 43], and other growth factors and signaling pathways are suggested to regulate the constitutive activation of this pathway . However, we were unable to test for mutations of PTEN, and there is a possibility that a mutation in PTEN was associated with the constitutive activation of Akt.
After cell injections into nude mice, HSA tumors developed from 4 cell lines. In these mice with developed tumors, no metastatic lesion was observed, similar to that of original canine HSA xenograft models . Similarly, metastatic tumor was not detected after subcutaneous injection of the human angiosarcoma cell line in nude mice despite tumorigenicity on the skin. Canine HSAs  as well as human angiosarcomas  have high metastatic biology that leads to poor prognosis; however, the established cell lines did not show these characteristics. Another study of a canine HSA cell line indicated that intravenous injection formed metastatic lesion in the lungs of SCID mice . The differences in the results of metastasis may depend on the route of cell injection or immunity of mice. Another possibility is that the metastatic property may be lost during passages of xenograft tumor or cell culture. However, immunohistochemical analysis in the present study revealed that the developed tumors after cell injection had high levels of phosphorylation of Akt at Ser473 and 4E-BP1 at Thr37/46 similar to that of the original cell lines. These in vivo models would be useful tools for evaluating the anti-tumor effect of inhibitors targeting the mTORC2/4E-BP1 pathway. Drugs targeting both mTORC2 and mTORC1 have been studied in acute myeloid leukemia and have shown marked anti-tumor effects . Because both mTORC1 and mTORC2 are activated during angiogenesis , mTORC1/mTORC2 inhibition may have a potent effect in HSA tumors through inhibition of not only tumor cell proliferation but also angiogenesis.
We have established 7 canine HSA cell lines from 3 xenograft canine HSAs. These cell lines showed diverse morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, and PDGF-B and their receptors. Cell proliferation was stimulated by these growth factors and FBS in one cell line, was stimulated by FBS alone in 3 cell lines, and was not stimulated by either growth factors or FBS in the remaining 3 cell lines. Phosphorylation of p44/42 Erk1/2 was increased in the presence of FBS in 4 cell lines and seemed to be related to serum-dependent proliferation. In contrast, phosphorylation of Akt at Ser473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 was not altered by FBS stimulation in 6 cell lines, suggesting that the mTORC2/Akt/4EBP1 pathway was constitutively activated in the present cell lines. After cell injection into nude mice, canine HSA tumors were formed in 4 cell lines. These tumors showed similar expression levels for phosphorylated Akt and 4E-BP1 as the original cell lines. Therefore, the present cell lines are useful models to investigate the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA in both in vitro and in vivo systems.
This study was supported by Grants-in-Aid for Scientific Research (C) Grant No. 23580439 from the Japan Society for the Promotion of Science.
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