Akt mediates an angiogenic switch in transformed keratinocytes
Carmen Segrelles,
Sergio Ruiz,
Mirentxu Santos,
Jesús Martínez-Palacio,
M. Fernanda Lara and
Jesús M. Paramio1
Department of Cell and Molecular Biology, CIEMAT, Av. Complutense 22, E-28040 Madrid, Spain
1 To whom correspondence should be addressed Email: jesusm.paramio{at}ciemat.es
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Abstract
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Akt signaling is involved in tumorigenesis via a number of different mechanisms that result in increased proliferation and decreased apoptosis. Previous data have demonstrated that Akt-mediated signaling is functionally involved in keratinocyte transformation. This work investigates the involvement of angiogenesis as a mediator of tumorigenesis in Akt-transformed keratinocytes. Tumors produced by subcutaneous injection of the latter showed increased angiogenic profiles associated with increased vascular endothelial growth factor (VEGF) protein levels. However, in contrast to v-rasHa-transformed keratinocytes, VEGF mRNA levels were not increased. The induction of VEGF protein by Akt is associated with increased phosphorylation and thus activation of p70S6K and eIF4E-binding protein 1, leading to increased VEGF translation. In addition, we observed increased metaloproteinases 2 and 9 expression, but not thrombospondin 1, in tumors derived from Akt-transformed keratinocytes. Collectively, these results demonstrate that Akt is an important mediator of angiogenesis in malignant keratinocytes through a post-transcriptional mechanism.
Abbreviations: HIF1
, hypoxia-inducible factor 1; MMP2, metaloproteinase 2; MMP9, metaloproteinase 9; TSP1, thrombospondin 1; TSP2 thrombospondin 2; VEGF, vascular endothelial growth factor
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Introduction
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The mouse skin carcinogenesis model has provided an important instrumental framework for understanding many of the concepts currently applied to human neoplasia (reviewed in refs 1,2). The importance of Akt-dependent signaling in mouse skin tumorigenesis has recently been demonstrated as Akt activity increases in parallel with the process of tumor progression and precedes that of MAPK/ERK (3). In addition, over-expression of Akt, which leads to increased Akt activity, exacerbates the tumorigenic behavior of murine keratinocytes (increased proliferation, decreased apoptosis and impaired differentiation) (3). In agreement, specific ablation of PTEN tumor suppressor gene in the epidermis leads to the formation of spontaneous epidermal tumors and increased sensitivity to chemical mouse skin carcinogenesis (4). Further, in experiments with transgenic mice, the ectopic expression of keratin K10which inhibits Akt activation (5,6)also results in dramatic inhibition of tumor development (5,7). These observations are in agreement with the current view that Akt functions in tumorigenesis in association with increased proliferation and survival of the transformed cells (8,9).
Besides alterations in proliferation and apoptosis, the induction of angiogenesis is essential in tumor growth since the generation of new vessels allows rapid tumor expansion and increases the likelihood of metastatic events. The acquisition of the angiogenic phenotype during tumorigenesis, the so-called angiogenic switch (10), is thought to be induced by a change in the balance of positive and negative regulators of endothelial cell growth (10). Among these, vascular endothelial growth factor (VEGF) is thought to be one of the major angiogenesis factors in malignant tumor growth (1113).
In the mouse skin carcinogenesis model, angiogenesis is an early event. The development of papillomas is preceded by a burst of angiogenesis (14). In addition, the activation of Ha-ras, the major critical event in tumor initiation in this system, plays a major role in the tumor angiogenic response inducing VEGF expression (15,16). The importance of VEGF in mouse skin carcinogenesis is demonstrated by accelerated tumor development in transgenic mice expressing VEGF (15), and by the rescue of tumor growth inhibition caused by functional EGFR abrogation promoted by VEGF expression (17). In addition to VEGF, other important factors such matrix metaloproteinases 2 (MMP2) and 9 (MMP9), thrombospondins 1 and 2 (TSP1, TSP2) have also recently emerged as important regulators of tumoral angiogenesis in mouse skin carcinogenesis (1824).
There is evidence that suggests the PI3K/PTEN/Akt pathway may be involved in tumor angiogenesis (reviewed in ref. 25). This appears to proceed mainly through the regulation of VEGF expression (2628) and TSP1 (29). However, the precise involvement of the different elements of the pathway and the molecular mechanisms resulting in the angiogenic response are not well understood. Given the reported essential role of Akt in mouse keratinocyte transformation (3), the aim of the present study was to investigate whether Akt signaling also regulated tumor angiogenesis in this system.
The results show that Akt modulates the angiogenic profile and VEGF up-regulation by a post-transcriptional mechanism associated with increased p70S6K and eIF4E-binding protein 1 (4E-BP1) phosphorylation. Further, Akt-induced tumors also show increased MMP2 and MMP9 expression, but no alteration in TSP1. This provides evidence that Akt plays a central role in the establishment of stromal changes leading to skin tumoral growth.
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Materials and methods
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Cell culture, transfection and in vivo tumorigenic assays
Mouse PB keratinocytes (30) and Akt-transfected derivatives (as 4080 pooled clones) were grown and subcutaneously injected into nu/nu mice as reported previously (3). In some cases, prior to subcutaneous injection, the cells were either mock- or retrovirus-infected as described previously (31). Helper-free retrovirus coding for v-rasHa was obtained from
2(ras) cells (32). Tumor growth was monitored with an external caliper for up to 7 weeks, measurements being made every 2 days [volume was calculated as
(4/3) x (width/2)2 x (length/2)]. One hour prior to death, mice were subcutaneously injected with BrdU (0.1 mg/g weight in 0.9% NaCl) to monitor proliferation. Tumor samples were excised and processed for histopathology, RNA or protein analysis. Similarly sized pieces from different tumors with similar stroma-tumor content, as determined by histopathology, and obtained at the same time points upon subcutaneous injection, were used in biochemical analyses. Proliferation and apoptosis measurements in the tumor samples were performed by double immunofluorescence using anti K5 (to detect tumor cells) and either anti BrdU mAb or TUNEL (Roche, Mannhein, Germany) essentially as described (3).
For luciferase assays plasmid pSV-Renilla was obtained from Promega and pVGEF-Luc KpnI (33) from ATCC. PB cells were transfected using Superfect (Qiagen) with pSV-Renilla, pVGEF-Luc KpnI and empty vector (pcDNA3) or plasmid coding from wt Akt or Ha-ras (Val12). Lysates were prepared 36 h after transfection and analyzed with the Dual Luciferase Reporter Assay system (Promega, Madison, WI). Relative luciferase expression was determined as the ratio of firefly to Renilla luciferase activity. Transfections were performed in triplicate, and the mean and standard error were calculated for each condition. PD98059, Wortmanin and rapamycin were purchased from Sigma (St Louis, MO) and used at 15 mM, 10 and 1 nM, respectively. These drugs were added to the transfected cells 24 h after transfection and the cultures were incubated for a further 24 h. At this time lysates were prepared and analyzed as above. Fluorescence-activated cell sorter (FACS) analysis was performed with methanol-fixed cells. DNA content was estimated with propidium iodide, and the cell cycle profile was analyzed by using multicycle software.
Immunohistochemical analysis of blood vessels and microvessel counting
Frozen tumor samples embedded in OCT (Tissue Tek, Sakura, Zoeterwoude, The Netherlands) and sectioned (6 µm thick) were fixed in acetone at 20°C for 5 min and the blood vessels visualized using rat anti-mouse CD31 (Pharmingen, diluted 1:30). To monitor vessel maturation, sections were also stained with smooth muscle
-actin using a mouse monoclonal (Sigma diluted 1/400). Microvessel counting was performed in the five areas of greatest vascularization in each tumor sample and expressed as an average. The area covered by the vessels and the mean vessel area was determined (using Microimage 4 software, Olympus, Silver Spring, MD) in the same fields screened for vessel counts after digitizing the images. For each type of tumor (either control or Akt), vessel counts and vessel-covered areas were recorded as the means of four to six individual tumor samples.
Northern blotting
Total RNA from different tumors or from pooled cultured clones (2040 different clones each) was isolated by the guanidine isothiocyanatephenolchloroform extraction and probed by northern blotting (15 µg/lane) for VEGF expression using mouse-specific VEGF probes as described previously (15). Equal loading was confirmed by hybridization with a 7S RNA probe. Quantification was performed using a Phosphorimager and Quantity One software (Bio-Rad, Hercules, CA).
Immunohistochemistry
The detection of CD·31,
-actin, MMP-2, MMP-9, VEGF and TSP1 was performed in frozen sections in parallel with antibodies reacting with mouse keratins (either rabbit polyclonal against K5 or mouse monoclonal against K14), essentially as described previously (3) using specific antibodies (see below). Secondary antibodies were purchased from Jackson Immunoresearch and used as described (3,5,6,34). Observations were made with a Zeiss Axiophot photomicroscope equipped with epifluorescence illumination and the correspondent filters to avoid cross channel contamination. At least four tumors of each type were analyzed. Controls omitting primary antibodies, or after the pre-incubation of the antibodies with the immunizing peptide (when available), were routinely performed.
Protein extracts and western blotting
Protein extracts were obtained from different tumors and used in western blotting (3). The following antibodies were used: Akt1 and 4E-BP1 (Santa Cruz Biotechnology), hypoxia-inducible factor 1 (HIF1
) (35,36) (Transduction Labs), phosphorylated Akt (Ser473; Cell Signaling, Beverly, MA), phosphorylated p70S6Kinase [Thr389 (37); Thr421/Thr424 (38); Cell Signaling], phosphorylated 4E-BP1 [Ser65; Cell Signaling (39)], MMP-2 (39), MMP-9 (41), VEGF (42) and TSP1 (43) (Neomarkers, Fremont, CA). WestPicoSignal (Pierce, Rockford, IL) was used to detect the bands according to the manufacturer's recommendations. Quantification was performed using Quantity One software (Bio-Rad).
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Results and discussion
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Akt induces tumorigenesis and tumor angiogenesis in keratinocytes
The PB keratinocyte cell line was obtained from a chemically induced mouse skin papilloma (30). However, it does not display increased EGFR expression nor mutations in Ha-ras gene (17,30). As a consequence, it is poorly tumorigenic in xenograft experiments, displaying long latency, reduced growth rate and the tumors obtained show highly differentiated phenotypes [Figure 1AD; see also (3,17,30)]. These characteristics, as potentially initiated cells, make this cell line a very useful model to analyze changes in possible increased tumorigenic properties upon a limited number of experimental alterations. In this regard, increased expression of Akt, leading to increased kinase activity [see below and (3,44)], promoted a dramatic enhancement in tumorigenic behavior, with reduced latency, increased growth rate and less differentiated phenotypes (Figure 1A, B, C' and D; see also ref. 3). Analysis of these tumors clearly demonstrated that Akt expression leads to increased proliferation and reduced apoptosis (Figure 1E) in agreement with our previous data (3). However, the increased proportion of cells in S phase upon Akt transfection in vitro (Figure 1F) does not seem to account for the growth rate observed in the tumors in vivo (Figure 1B) and suggests the existence of other mechanisms that support tumor development.

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Fig. 1. Increased tumorigenic potential of Akt-transfected keratinocytes. Pooled clones (2040) of PB keratinocytes after transfection with pcDNA3 (open squares) or wt Akt (closed squares) were injected subcutaneously into nude mice. The time of appearance (A) and the mean volume of tumors (B) were subsequently monitored. (C and D) Histological appearance of tumors from control keratinocytes showing well-differentiated morphology (C), whereas Akt transfection leads to poorly differentiated (C' and D) morphologies. (E) Increased proliferation and decreased apoptosis in tumors generated by Akt-transfected PB keratinocytes. The proliferation of tumors (closed bars) was analyzed by the ability of the cells to incorporate BrdU, whereas apoptosis induction was measured by TUNEL labeling (open bars). At least six fields per tumor and five tumors of each type were scored for each analysis. Data are shown as mean ± SD. (F) Percentage of Akt- and pcDNA-transfected pooled clones in S-phase in vitro analyzed by FACS after being stained with propidium iodide.
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The induction of angiogenesis is essential in tumor growth since the generation of new vessels allows rapid tumor expansion providing the environment necessary to allow the unrestrained growth of tumor cells and to prevent necrosis and correlates with aggressiveness. The angiogenic switch from vascular quiescence to up-regulation of angiogenesis has been observed in the early stages of skin carcinogenesis (14). Our previous data demonstrating increased Akt activation in parallel with the process of tumor progression (3) could suggest that such early Akt activation might also parallel angiogenesis. In agreement, we observed a reddish appearance in Akt-induced tumors, compared with a pale aspect of control tumors (Figure 2A).

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Fig. 2. Increased angiogenic aspect of Akt-induced tumors. (A) Appearance of tumors upon subcutaneous injection of pcDNA3 (right flank) or Akt-transfected keratinocytes (left flank). Note the reddish appearance of the Akt-induced tumors. (B and B') Representative CD31 immunostaining (green) of tumors from control (B) and Akt-transfected keratinocytes (B') showing increased number of more mature vessels in tumors from Akt-transfected PB cells. (C and C') Detection of smooth muscle -actin (green) in tumors from control (C) and Akt-transfected keratinocytes. Note that only small narrow vessels displayed positive reactivity in the control tumors. Keratin K5 (red in B, B', C and C') was used to counterstain tumoral cells. Arrows in (B) denote lacunar blood vessels commonly observed in control tumors. Bars = 100 µm.
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To confirm the possible changes in angiogenesis we analyzed the tumor-associated vascularization. Frozen sections were stained for the endothelial junction molecule CD31 (14). In control tumor samples, the vessels appeared clear and lacunar (Figure 2B, arrows), similar to those observed in papillomas and highly differentiated squamous carcinomas and suggestive of immaturity and poor functionality (14). On the contrary, Akt-induced tumors showed an increased number of blood vessels, which appeared also to be narrower than those of controls (Figure 2B') and similar to those observed in poorly differentiated carcinomas, which have been associated with a more mature and functional stage (14). To verify this suggestion, double labeling against keratin (to label tumor cells) and smooth muscle
actin was performed. This is a well-known marker of mature vessels (45,46). The results (Figure 2C and C') confirm that the tumors generated by Akt-transfected keratinocytes show a higher grade of blood vessel maturation compared with controls. To further substantiate these observations in a quantitative manner, computer-assisted morphometric image analysis was performed in the CD31 stained sections. This revealed that both vessel density and the relative area occupied by tumor blood vessels were increased in Akt-derived tumors compared with control samples (Figure 3A and B). On the other hand, the mean size of the vessels was larger in control than in Akt-induced tumors (Figure 3C). Collectively, these data show that Akt induces an angiogenic switch in keratinocytes.

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Fig. 3. Histomorphometric analysis of blood vessels of different tumors. Computer-assisted analysis of blood vessels following CD31 immunostaining showing increased numbers of vessels (A) covering major areas (B) and their narrow and smaller size (C) in Akt-derived tumors compared with control samples. At least five tumors of each type were analyzed using four to six fields on each. Data are shown as mean ± SD.
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Akt-induced angiogenesis is not mediated by increased VEGF mRNA
VEGF mediates the essential angiogenic response required for mouse skin tumor progression (1517). VEGF expression can be regulated at the transcriptional and mRNA stability levels, leading to increased VEGF mRNA steady state (47,48). To investigate if Akt can drive VEGF mRNA expression, mRNAs extracted from several control and Akt-derived tumors obtained at the same time and containing similar tumorstroma ratio were used in northern blot analysis. No significant differences were seen among the different samples (Figure 4A and A'). To substantiate this observation, the VEGF mRNA levels were also analyzed in pooled clones from control or Akt-transfected PB cells prior to the generation of subcutaneous tumors (Figure 4B and B'). Again, we did not observe increased expression of VEGF mRNA by Akt transfection. Finally, as a positive control and given the reported induction of VEGF mRNA by activated Ha-ras (15) we monitored VEGF expression in tumors upon v-rasHa expression. Empty vector and Akt-transfected PB keratinocytes were transduced with a v-rasHa-coding retrovirus. The mock or v-rasHa infected cells were subsequently used in subcutaneous injection experiments and VEGF mRNA expression was then analyzed in four types of tumor (control, control plus v-rasHa, Akt and Akt plus v-rasHa). The obtained results (Figure 4C and C') confirmed the induction of VEGF mRNA expression mediated by ras, in agreement with the previous reported data in mouse keratinocytes in vivo and in vitro (15). On the other hand, such increases were similar in all cases, irrespective of whether the cells were empty vector- or Akt-transfected keratinocytes (Figure 4C). Collectively, these data indicated that Akt is not involved in VEGF mRNA expression in PB keratinocytes.

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Fig. 4. Akt does not induce VEGF mRNA. Total mRNA from different tumor samples (A and C) or pooled cell clones prior to subcutaneous injection (B) was probed for the expression of VEGF by northern blotting. To control loading, a 7S probe was used in all the cases. Note that no increase in VEGF expression was observed in Akt-derived samples, whereas v-rasHa infection promoted the increased VEGF mRNA expression both in control and Akt-derived tumors (C). (A'C') Semi-quantitative analysis obtained by densitometry of the corresponding northern blot data. (D) Luciferase activity from the VEGF promoter in PB cells co-transfected with pcDNA3, Akt or Ha-ras (Val12). Note that only Ha-ras (Val12) expression leads to significant increase in VEGF promoter activity. (D) Luciferase activity from the VEGF promoter in PB cells co-transfected with Ha-ras (Val12) and treated for 24 h with the stated inhibitors. Data in (D and D') come from three independent experiments and are shown as mean ± SD.
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To further support this observation we monitored the activity of the VEGF promoter (33) in control or Akt-transfected PB keratinocytes. To this we co-transfected Akt, ras or empty vector (pcDNA3) together with a reporter plasmid coding for firefly luciferase under the control of mouse VEGF promoter (33) and the activity of this latter was analyzed. The obtained data confirmed that ras, but not Akt induces the transcription of VEGF gene in PB keratinocytes (Figure 4D). The present observations disagree with the previously reported involvement of the PI3K/Akt pathway in VEGF mRNA expression (28,49). This could be due to a cell type-specific effect similar to those reported previously in fibroblast and some epithelial cell lines (50), or to a major effect of other alternative pathways. In this regard it has been shown that VEGF mRNA is induced by TPA treatment in mouse skin (15) by the p38-dependent pathway in breast cancer cells (51), and through both MAPK/ERK and PI3K/Akt in human squamous cell carcinoma cells (52). Although at present it cannot be said which of these are responsible for VEGF mRNA up-regulation, the observation that Ha-ras induces VEGF mRNA in parental or Akt-transfected keratinocytes (Figure 4) strongly suggests that other ras-dependent pathways (53,54) different to that of Akt activation are involved. In this regard, experiments using drugs that specifically block particular signaling pathways indicate that inhibition of ERK and to a lesser extent PI-3K or mTOR, leads to a substantial reduction in the transcription of VEGF gene in PB keratinocytes (Figure 4D'), thus suggesting that in our experimental settings multiple Ha-ras-dependent pathways converge to increase the VEGF transcription.
Akt induces VEGF, MMP2 and MMP9 protein expression
The expression of VEGF is regulated at many levels by disparate stimuli. In addition to mRNA steady state levels, VEGF regulation can also occur at the level of VEGF translation (5558). Consequently, we monitored the expression of VEGF protein in parallel with Akt and active-phosphorylated-Akt (P-Ser 473) in extracts from control- and Akt-derived tumors. In agreement with previous results (3,44), the tumors derived from Akt-transfected keratinocytes also showed increased active Akt (Figure 5A and A'). In addition, the expression of VEGF protein in tumors derived from Akt-transfected keratinocytes was increased (Figure 5A), while no increase in HIF1
protein expression was seen (Figure 5A). Given that the transcriptional activation of VEGF is mediated mainly by this transcription factor, the absence of increased HIF1
protein levels might correspond to the lack of induction of VEGF mRNA observed (Figure 4). The transcriptional activation of VEGF is mediated mainly by the transcription factor HIF1
. The present data, which show no increase in HIF1
protein (Figure 5A and A'), are therefore in agreement with the lack of change in VEGF mRNA in cells and tumors (Figure 4).

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Fig. 5. Akt induces VEGF, MMP2 and MMP9 proteins. (A) Protein extracts from different tumors obtained with Akt-transfected cells and from control cells [including well-differentiated (w-d) and poorly differentiated (p-d)] were probed by western blotting with the quoted antibodies. Note that increased Akt protein leads to increased active Akt levels and also to increased VEGF (A), MMP2 and MMP9 (C) protein levels. Of note also is the increased phosphorylation of p70S6K and 4E-BP1 protein promoted by this increase in Akt activity (B). No significant alterations in the protein levels of HIF1 (A) and TSP-1 (C) were observed. Blots were reprobed with an antibody against tubulin as a loading control. (A'C') Semi-quantitative analysis obtained by densitometry of the corresponding western blot data.
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The finding that Akt regulates the translation of VEGF prompted an investigation of the possible mechanism underlying this effect. It has been shown that the VEGF contains an unusually long and structured 5' untranslated region (UTR) (59). VEGF mRNA translation can take place by an internal ribosome entry process (56,57,6062). In addition, VEGF mRNA translation can be regulated by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex (58,63,64). By binding the cap structure, eIF-4E recruits mRNAs to the eIF-4F complex to enable ribosome loading. mRNAs harboring lengthy, highly structured 5' UTRs, is suppressed except when eIF-4E is engaged with the eIF-4F complex. In addition, this complex is further regulated by translational repressors, the 4E-BPs. It has been reported that Akt can mediate the phosphorylation of 4E-BP1 disrupting its binding to eIF-4E (65). Furthermore, 4E-BP1 is co-ordinately regulated with p70S6K in many circumstances, and the p70S6 kinase is also activated by an Akt-dependent pathway (66,67). We consequently hypothesized that Akt might regulate VEGF expression through increased translation. The phosphorylation of 4E-BP1 and p70S6 kinase was thus assessed in different tumors derived from control and Akt-transfected keratinocytes. A marked increase in 4E-BP1 (on Ser 65) and p70S6K (on Thr389) phosphorylation was evident in tumors derived from Akt-transfected keratinocytes compared with those derived from control keratinocytes (Figure 5B and B'). On the other hand, similar levels of total 4E-BP1 and p70S6K proteins were observed, as well as phosphorylation of p70S6K on Thr421/424 (which is dependent on the raf/MAPK cascade), irrespective of Akt activity (Figure 5B and B').
At present we may not discern, whether eIF4 or p70S6K-dependent mechanism is responsible for the induced VEGF translation. In this regard, the presence of a prototype for the terminal oligopyrimidine (TOP), whose expression is controlled by the activity of p70S6K, in the VEGF mRNA has not been demonstrated, thus suggesting a major involvement of 4E-BP1 phosphorylation. However, the fact that p70S6K is more sensitive to down-regulation by glucocorticoids than is 4E-BP1 (66,67), and glucocorticoids can repress skin tumor development by preventing Akt activation (68,69) may suggest a preponderant role of p70S6K. These aspects will be studied in the near future.
On the other hand, both p706SK and 4E-BP1 phosphorylation are rapamycin sensitive (65,7074), thus suggesting the involvement of mTOR in the increased VEGF protein translation. In agreement, preliminary experiments indicate that rapamycin treatment decreases tumorigenic behavior of Akt and Ha-ras transduced keratinocytes (Segrelles et al., unpublished results). Although the potential involvement of anti-angiogenic process has not been yet tested, these data indicate that mTOR acts downstream of Akt and Ha-ras in keratinocyte transformation. Collectively, these data indicate that the increase in VEGF can be attributed to the ability of Akt to regulate the mTOR signaling pathway, which may account for increased VEGF translation. On the other hand, HIF1
expression has also been described to be under the control of the PI3K/PTEN/Akt/mTOR pathway (7577). Given the increased Akt activity in the tumors (Figure 5, see also refs 3,44), the lack of increased levels of HIF1
might be due to hypoxic conditions irrespective of tumor origin. Indeed, it has been reported that hypoxia-dependent induction of HIF1
is moderately affected by inhibitors of this pathway (36,75,78,79).
The process of tumor angiogenesis is not only dependent on VEGF production but rather on the balance of positive and negative regulators of endothelial cell growth (10). Among these factors, and besides VEGF, MMP2 and MMP9, are well-recognized inducers of tumor angiogenesis (19,20,8082), whereas TSP1 is considered an inhibitor (2023,83). Their expression was therefore monitored in different tumors derived from control or Akt-transfected keratinocytes. The results indicate that Akt induced the expression of MMP2 and MMP9 (Figure 5C and C') in agreement with previous data (18,8486). On the other hand we found that TSP-1 levels were similar in the different samples (Figure 5C and C') indicating that TSP-1 levels are not modulated by Akt. These results seemed to be at variance with the reported activity of PTEN to induce TSP1 expression (29). However, it is worth noting that PTEN may influence other PI3K-dependent pathways besides the activation of Akt. Therefore, our data would indicate that TSP-1 expression depends on these alternate pathways. In this regard, it has recently been demonstrated that the activation of Rho mediated by increased PI3K activity is responsible for the down-regulation of TSP-1 mediated by ras in mammary and kidney cells (87).
Finally, to further substantiate these observations, the expression of these molecules in parallel with VEGF was studied immunohistochemically in tumors derived from subcutaneous injections of Akt-transfected and control PB keratinocytes. In agreement with the western data (Figure 5), Akt led to increased VEGF, MMP2 and MMP9 expression (Figure 6A', B' and C') compared with control samples (Figure 6A, B and C). Interestingly, the expression of MMP2 and MMP9 was found mainly in the tumor periphery and few tumor cells, either Akt or v-rasHa (Figure 6B', B'', C' and C'', respectively) also display immunoreactivity against these molecules, thus suggesting that tumor cells are not the primary source of these MMP. On the other hand, no changes in TSP1 expression were observed between control and Akt-derived tumors (Figure 5D and D'), whereas v-rasHa transduction led to decreased expression (Figure 5D'').

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Fig. 6. Immunohistochemical detection of VEGF, MMP2 and TSP1 in tumors. Frozen sections of tumors obtained after subcutaneous injection of parental (AD), Akt-transfected (A'D'), and control keratinocytes upon v-rasHa infection (A''D''), were stained for VEGF (green in A, A' and A''), MMP2 (green in B, B' and B''), MMP9 (green in C, C' and C'') and TSP-1 (green in D, D' and D''). Together with K14 or K5 (in red) to denote the tumoral cells. Note that the Akt and v-rasHa infection led to increased VEGF, MMP2 and MMP9 protein expression, whereas only v-rasHa infection decreased TSP1 expression. In addition, MMP2 and MMP9 immunoreactivity is predominant at the tumor boundary (B', B'', C' and C''). Bar = 50 µm.
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Collectively the results presented here demonstrate that Akt is an important mediator of angiogenesis in keratinocytes, leading to an increased number of blood vessels with a more mature appearance. This seemed to proceed through a post-transcriptional process due to the activation of mTOR signaling leading to increased VEGF levels. These allow the neovascularization of the tumors allowing the recruitment of other cell types that then may secrete other molecules such as MMP2 and MMP9 (83,88) relevant for the angiogenic switch. In addition, present data imply that drugs that reduce Akt or Akt kinase activity, or which inhibit Akt-mediated translation increase, could be of crucial clinical interest to modulate angiogenic profile in human cancers.
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Acknowledgments
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We thank F.Larcher for providing VEGF probes and critical reading of the manuscript, the animal facility personnel of the CIEMAT for the excellent care of the animals, and I.de los Santos and P.Hernández for their work involving the histological preparations. This work was partially supported by grant SAF2002-01037 from the MCYT and 08.1/0054/2001.1 from the CAM (to J.M.P.).
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References
|
---|
- Slaga,T.J., Budunova,I.V., Gimenez-Conti,I.B. and Aldaz,C.M. (1996) The mouse skin carcinogenesis model. J. Invest. Dermatol. Symp. Proc., 1, 151156.
- Yuspa,S.H., Dlugosz,A.A., Denning,M.F. and Glick,A.B. (1996) Multistage carcinogenesis in the skin. J. Invest. Dermatol. Symp. Proc., 1, 147150.
- Segrelles,C., Ruiz,S., Perez,P. et al. (2002) Functional roles of Akt signaling in mouse skin tumorigenesis. Oncogene, 21, 5364.[CrossRef][ISI][Medline]
- Suzuki,A., Itami,S., Ohishi,M. et al. (2003) Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation. Cancer Res., 63, 674681.[Abstract/Free Full Text]
- Santos,M., Paramio,J.M., Bravo,A., Ramirez,A. and Jorcano,J.L. (2002) The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis. J. Biol. Chem., 277, 1912219130.[Abstract/Free Full Text]
- Paramio,J.M., Segrelles,C., Ruiz,S. and Jorcano,J.L. (2001) Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest. Mol. Cell. Biol., 21, 74497459.[Abstract/Free Full Text]
- Santos,M., Ballestin,C., Garcia-Martin,R. and Jorcano,J.L. (1997) Delays in malignant tumor development in transgenic mice by forced epidermal keratin 10 expression in mouse skin carcinomas. Mol. Carcinog., 20, 39.[CrossRef][ISI][Medline]
- Nicholson,K.M. and Anderson,N.G. (2002) The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal., 14, 381395.[CrossRef][ISI][Medline]
- Brazil,D.P. and Hemmings,B.A. (2001) Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci., 26, 657664.[CrossRef][ISI][Medline]
- Hanahan,D. and Folkman,J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86, 353364.[ISI][Medline]
- Conti,C.J. (2002) Vascular endothelial growth factor: regulation in the mouse skin carcinogenesis model and use in antiangiogenesis cancer therapy. Oncologist, 7, 411.[Abstract/Free Full Text]
- Ferrara,N. (2002) VEGF and the quest for tumour angiogenesis factors. Nat. Rev. Cancer, 2, 795803.[CrossRef][ISI][Medline]
- Harmey,J.H. and Bouchier-Hayes,D. (2002) Vascular endothelial growth factor (VEGF), a survival factor for tumour cells: implications for anti-angiogenic therapy. Bioessays, 24, 280283.[CrossRef][ISI][Medline]
- Bolontrade,M.F., Stern,M.C., Binder,R.L., Zenklusen,J.C., Gimenez-Conti,I.B. and Conti,C.J. (1998) Angiogenesis is an early event in the development of chemically induced skin tumors. Carcinogenesis, 19, 21072113.[Abstract]
- Larcher,F., Robles,A.I., Duran,H., Murillas,R., Quintanilla,M., Cano,A., Conti,C.J. and Jorcano,J.L. (1996) Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels. Cancer Res., 56, 53915396.[Abstract]
- Larcher,F., Murillas,R., Bolontrade,M., Conti,C.J. and Jorcano,J.L. (1998) VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development. Oncogene, 17, 303311.[CrossRef][ISI][Medline]
- Casanova,M.L., Larcher,F., Casanova,B., Murillas,R., Fernandez-Acenero,M.J., Villanueva,C., Martinez-Palacio,J., Ullrich,A., Conti,C.J. and Jorcano,J.L. (2002) A critical role for ras-mediated, epidermal growth factor receptor-dependent angiogenesis in mouse skin carcinogenesis. Cancer Res., 62, 34023407.[Abstract/Free Full Text]
- Park,B.K., Zeng,X. and Glazer,R.I. (2001) Akt1 induces extracellular matrix invasion and matrix metalloproteinase-2 activity in mouse mammary epithelial cells. Cancer Res., 61, 76477653.[Abstract/Free Full Text]
- Papathoma,A.S., Zoumpourlis,V., Balmain,A. and Pintzas,A. (2001) Role of matrix metalloproteinase-9 in progression of mouse skin carcinogenesis. Mol. Carcinog., 31, 7482.[CrossRef][ISI][Medline]
- Bergers,G., Brekken,R., McMahon,G. et al. (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell. Biol., 2, 737744.[CrossRef][ISI][Medline]
- Bleuel,K., Popp,S., Fusenig,N.E., Stanbridge,E.J. and Boukamp,P. (1999) Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc. Natl Acad. Sci. USA, 96, 20652070.[Abstract/Free Full Text]
- Hawighorst,T., Oura,H., Streit,M., Janes,L., Nguyen,L., Brown,L.F., Oliver,G., Jackson,D.G. and Detmar,M. (2002) Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice. Oncogene, 21, 79457956.[CrossRef][ISI][Medline]
- Streit,M., Velasco,P., Brown,L.F., Skobe,M., Richard,L., Riccardi,L., Lawler,J. and Detmar,M. (1999) Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am. J. Pathol., 155, 441452.[Abstract/Free Full Text]
- Hawighorst,T., Velasco,P., Streit,M., Hong,Y.K., Kyriakides,T.R., Brown,L.F., Bornstein,P. and Detmar,M. (2001) Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism. EMBO J., 20, 26312640.[Abstract/Free Full Text]
- Shiojima,I. and Walsh,K. (2002) Role of Akt signaling in vascular homeostasis and angiogenesis. Circ. Res., 90, 12431250.[Abstract/Free Full Text]
- Zundel,W., Schindler,C., Haas-Kogan,D. et al. (2000) Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev., 14, 391396.[Abstract/Free Full Text]
- Huang,J. and Kontos,C.D. (2002) PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J. Biol. Chem., 277, 1076010766.[Abstract/Free Full Text]
- Jiang,B.H., Zheng,J.Z., Aoki,M. and Vogt,P.K. (2000) Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc. Natl Acad. Sci. USA, 97, 17491753.[Abstract/Free Full Text]
- Wen,S., Stolarov,J., Myers,M.P., Su,J.D., Wigler,M.H., Tonks,N.K. and Durden,D.L. (2001) PTEN controls tumor-induced angiogenesis. Proc. Natl Acad. Sci. USA, 98, 46224627.[Abstract/Free Full Text]
- Yuspa,S.H., Morgan,D., Lichti,U., Spangler,E.F., Michael,D., Kilkenny,A. and Hennings,H. (1986) Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol. Carcinogenesis, 7, 949958.[Abstract]
- Paramio,J.M., Segrelles,C., Ruiz,S., Martin-Caballero,J., Page,A., Martinez,J., Serrano,M. and Jorcano,J.L. (2001) The ink4a/arf tumor suppressors cooperate with p21cip1/waf in the processes of mouse epidermal differentiation, senescence and carcinogenesis. J. Biol. Chem., 276, 4420344211.[Abstract/Free Full Text]
- Roop,D.R., Lowy,D.R., Tambourin,P.E., Strickland,J., Harper,J.R., Balaschak,M., Spangler,E.F. and Yuspa,S.H. (1986) An activated Harvey ras oncogene produces benign tumours on mouse epidermal tissue. Nature, 323, 822824.[ISI][Medline]
- Forsythe,J.A., Jiang,B.H., Iyer,N.V., Agani,F., Leung,S.W., Koos,R.D. and Semenza,G.L. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol., 16, 46044613.[Abstract]
- Santos,M., Perez,P., Segrelles,C., Ruiz,S., Jorcano,J.L. and Paramio,J.M. (2003) Impaired NF-kappa B activation and increased production of tumor necrosis factor alpha in transgenic mice expressing keratin K10 in the basal layer of the epidermis. J. Biol. Chem., 278, 1342213430.[Abstract/Free Full Text]
- Srinivas,V., Leshchinsky,I., Sang,N., King,M.P., Minchenko,A. and Caro,J. (2001) Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway. J. Biol. Chem., 276, 2199521998.[Abstract/Free Full Text]
- Arsham,A.M., Plas,D.R., Thompson,C.B. and Simon,M.C. (2002) Phosphatidylinositol 3-kinase/Akt signaling is neither required for hypoxic stabilization of HIF-1 alpha nor sufficient for HIF-1-dependent target gene transcription. J. Biol. Chem., 277, 1516215170.[Abstract/Free Full Text]
- Weng,Q.P., Kozlowski,M., Belham,C., Zhang,A., Comb,M.J. and Avruch,J. (1998) Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem., 273, 1662116629.[Abstract/Free Full Text]
- Pullen,N. and Thomas,G. (1997) The modular phosphorylation and activation of p70s6k. FEBS Lett., 410, 7882.[CrossRef][ISI][Medline]
- Aoki,M., Blazek,E. and Vogt,P.K. (2001) A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc. Natl Acad. Sci. USA, 98, 136141.[Abstract/Free Full Text]
- Margulies,I.M., Hoyhtya,M., Evans,C., Stracke,M.L., Liotta,L.A. and Stetler-Stevenson,W.G. (1992) Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma. Cancer Epidemiol. Biomarkers Prev., 1, 467474.[Abstract]
- Nikkari,S.T., Hoyhtya,M., Isola,J. and Nikkari,T. (1996) Macrophages contain 92-kd gelatinase (MMP-9) at the site of degenerated internal elastic lamina in temporal arteritis. Am. J. Pathol., 149, 14271433.[Abstract]
- Boocock,C.A., Charnock-Jones,D.S., Sharkey,A.M., McLaren,J., Barker,P.J., Wright,K.A., Twentyman,P.R. and Smith,S.K. (1995) Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma. J. Natl Cancer Inst., 87, 506516.[Abstract]
- Dixit,V.M., Galvin,N.J., O'Rourke,K.M. and Frazier,W.A. (1986) Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes. J. Biol. Chem., 261, 19621968.[Medline]
- Leis,H., Segrelles,C., Ruiz,S., Santos,M. and Paramio,J.M. (2002) Expression, localization and activity of glycogen synthase kinase 3beta during mouse skin tumorigenesis. Mol. Carcinog., 35, 180185.[CrossRef][ISI][Medline]
- Huang,J., Frischer,J.S., Serur,A. et al. (2003) Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc. Natl Acad. Sci. USA, 100, 77857790.[Abstract/Free Full Text]
- Hungerford,J.E., Owens,G.K., Argraves,W.S. and Little,C.D. (1996) Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev. Biol., 178, 375392.[CrossRef][ISI][Medline]
- von Marschall,Z., Cramer,T., Hocker,M., Finkenzeller,G., Wiedenmann,B. and Rosewicz,S. (2001) Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma. Gut, 48, 8796.[Abstract/Free Full Text]
- Nabors,L.B., Gillespie,G.Y., Harkins,L. and King,P.H. (2001) HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs. Cancer Res., 61, 21542161.[Abstract/Free Full Text]
- Mazure,N.M., Chen,E.Y., Laderoute,K.R. and Giaccia,A.J. (1997) Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. Blood, 90, 33223331.[Abstract/Free Full Text]
- Rak,J., Mitsuhashi,Y., Sheehan,C., Tamir,A., Viloria-Petit,A., Filmus,J., Mansour,S.J., Ahn,N.G. and Kerbel,R.S. (2000) Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res., 60, 490498.[Abstract/Free Full Text]
- Xiong,S., Grijalva,R., Zhang,L., Nguyen,N.T., Pisters,P.W., Pollock,R.E. and Yu,D. (2001) Up-regulation of vascular endothelial growth factor in breast cancer cells by the heregulin-beta1-activated p38 signaling pathway enhances endothelial cell migration. Cancer Res., 61, 17271732.[Abstract/Free Full Text]
- Dong,G., Chen,Z., Li,Z.Y., Yeh,N.T., Bancroft,C.C. and Van Waes,C. (2001) Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleukin-8 and vascular endothelial growth factor in head and neck squamous cell carcinoma. Cancer Res., 61, 59115918.[Abstract/Free Full Text]
- Campbell,S.L., Khosravi-Far,R., Rossman,K.L., Clark,G.J. and Der,C.J. (1998) Increasing complexity of Ras signaling. Oncogene, 17, 13951413.[CrossRef][ISI][Medline]
- Vojtek,A.B. and Der,C.J. (1998) Increasing complexity of the Ras signaling pathway. J. Biol. Chem., 273, 1992519928.[Free Full Text]
- Kevil,C.G., De Benedetti,A., Payne,D.K., Coe,L.L., Laroux,F.S. and Alexander,J.S. (1996) Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis. Int. J. Cancer, 65, 785790.[CrossRef][ISI][Medline]
- Akiri,G., Nahari,D., Finkelstein,Y., Le,S.Y., Elroy-Stein,O. and Levi,B.Z. (1998) Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription. Oncogene, 17, 227236.[CrossRef][ISI][Medline]
- Stein,I., Itin,A., Einat,P., Skaliter,R., Grossman,Z. and Keshet,E. (1998) Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia. Mol. Cell. Biol., 18, 31123119.[Abstract/Free Full Text]
- Chung,J., Bachelder,R.E., Lipscomb,E.A., Shaw,L.M. and Mercurio,A.M. (2002) Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J. Cell. Biol., 158, 165174.[Abstract/Free Full Text]
- Shima,D.T., Kuroki,M., Deutsch,U., Ng,Y.S., Adamis,A.P. and D'Amore,P.A. (1996) The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit and characterization of transcriptional and post-transcriptional regulatory sequences. J. Biol. Chem., 271, 38773883.[Abstract/Free Full Text]
- Huez,I., Bornes,S., Bresson,D., Creancier,L. and Prats,H. (2001) New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation. Mol. Endocrinol., 15, 21972210.[Abstract/Free Full Text]
- Huez,I., Creancier,L., Audigier,S., Gensac,M.C., Prats,A.C. and Prats,H. (1998) Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA. Mol. Cell. Biol., 18, 61786190.[Abstract/Free Full Text]
- Miller,D.L., Dibbens,J.A., Damert,A., Risau,W., Vadas,M.A. and Goodall,G.J. (1998) The vascular endothelial growth factor mRNA contains an internal ribosome entry site. FEBS Lett., 434, 417420.[CrossRef][ISI][Medline]
- Scott,P.A., Smith,K., Poulsom,R., De Benedetti,A., Bicknell,R. and Harris,A.L. (1998) Differential expression of vascular endothelial growth factor mRNA vs protein isoform expression in human breast cancer and relationship to eIF-4E. Br. J. Cancer, 77, 21202128.[ISI][Medline]
- Graff,J.R. and Zimmer,S.G. (2003) Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs. Clin. Exp. Metastasis, 20, 265273.[CrossRef][ISI][Medline]
- Gingras,A.C., Kennedy,S.G., O'Leary,M.A., Sonenberg,N. and Hay,N. (1998) 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt (PKB) signaling pathway. Genes Dev., 12, 502513.[Abstract/Free Full Text]
- Goncharova,E.A., Goncharov,D.A., Eszterhas,A. et al. (2002) Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J. Biol. Chem., 277, 3095830967.[Abstract/Free Full Text]
- Shi,Y., Hsu,J.H., Hu,L., Gera,J. and Lichtenstein,A. (2002) Signal pathways involved in activation of p70S6K and phosphorylation of 4E-BP1 following exposure of multiple myeloma tumor cells to interleukin-6. J. Biol. Chem., 277, 1571215720.[Abstract/Free Full Text]
- Leis,H., Page,A., Ramirez,A., Bravo,A., Segrelles,C., Paramio,J., Barettino,D., Jorcano,J.L. and Perez,P. (2004) Glucocorticoid receptor counteracts tumorigenic activity of Akt in skin through interference with the phosphatidylinositol 3-kinase signaling pathway. Mol. Endocrinol., 18, 303311.[Abstract/Free Full Text]
- Budunova,I.V., Kowalczyk,D., Perez,P., Yao,Y.J., Jorcano,J.L. and Slaga,T.J. (2003) Glucocorticoid receptor functions as a potent suppressor of mouse skin carcinogenesis. Oncogene, 22, 32793287.[CrossRef][ISI][Medline]
- Neshat,M.S., Mellinghoff,I.K., Tran,C., Stiles,B., Thomas,G., Petersen,R., Frost,P., Gibbons,J.J., Wu,H. and Sawyers,C.L. (2001) Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl Acad. Sci. USA, 98, 1031410319.[Abstract/Free Full Text]
- Podsypanina,K., Lee,R.T., Politis,C. et al. (2001) An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/ mice. Proc. Natl Acad. Sci. USA, 98, 1032010325.[Abstract/Free Full Text]
- Sekulic,A., Hudson,C.C., Homme,J.L., Yin,P., Otterness,D.M., Karnitz,L.M. and Abraham,R.T. (2000) A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res., 60, 35043513.[Abstract/Free Full Text]
- Raught,B. and Gingras,A.C. (1999) eIF4E activity is regulated at multiple levels. Int. J. Biochem. Cell Biol., 31, 4357.[CrossRef][ISI][Medline]
- Nomura,M., He,Z., Koyama,I., Ma,W.Y., Miyamoto,K. and Dong,Z. (2003) Involvement of the Akt/mTOR pathway on EGF-induced cell transformation. Mol. Carcinog., 38, 2532.[CrossRef][ISI][Medline]
- Zhong,H., Chiles,K., Feldser,D., Laughner,E., Hanrahan,C., Georgescu,M.M., Simons,J.W. and Semenza,G.L. (2000) Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res., 60, 15411545.[Abstract/Free Full Text]
- Jiang,B.H., Jiang,G., Zheng,J.Z., Lu,Z., Hunter,T. and Vogt,P.K. (2001) Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ., 12, 363369.[Abstract/Free Full Text]
- Treins,C., Giorgetti-Peraldi,S., Murdaca,J., Semenza,G.L. and Van Obberghen,E. (2002) Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J. Biol. Chem., 277, 2797527981.[Abstract/Free Full Text]
- Blancher,C., Moore,J.W., Robertson,N. and Harris,A.L. (2001) Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway. Cancer Res., 61, 73497355.[Abstract/Free Full Text]
- Alvarez-Tejado,M., Alfranca,A., Aragones,J., Vara,A., Landazuri,M.O. and del Peso,L. (2002) Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J. Biol. Chem., 277, 1350813517.[Abstract/Free Full Text]
- Coussens,L.M., Tinkle,C.L., Hanahan,D. and Werb,Z. (2000) MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell, 103, 481490.[ISI][Medline]
- Silletti,S., Kessler,T., Goldberg,J., Boger,D.L. and Cheresh,D.A. (2001) Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo. Proc. Natl Acad. Sci. USA, 98, 119124.[Abstract/Free Full Text]
- Pfeifer,A., Kessler,T., Silletti,S., Cheresh,D.A. and Verma,I.M. (2000) Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2. Proc. Natl Acad. Sci. USA, 97, 1222712232.[Abstract/Free Full Text]
- Coussens,L.M., Raymond,W.W., Bergers,G., Laig-Webster,M., Behrendtsen,O., Werb,Z., Caughey,G.H. and Hanahan,D. (1999) Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev., 13, 13821397.[Abstract/Free Full Text]
- Kubiatowski,T., Jang,T., Lachyankar,M.B., Salmonsen,R., Nabi,R.R., Quesenberry,P.J., Litofsky,N.S., Ross,A.H. and Recht,L.D. (2001) Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas. J. Neurosurg., 95, 480488.[ISI][Medline]
- Kim,D., Kim,S., Koh,H., Yoon,S.O., Chung,A.S., Cho,K.S. and Chung,J. (2001) Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J., 15, 19531962.[Abstract/Free Full Text]
- Thant,A.A., Nawa,A., Kikkawa,F., Ichigotani,Y., Zhang,Y., Sein,T.T., Amin,A.R. and Hamaguchi,M. (2000) Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells. Clin. Exp. Metastasis, 18, 423428.[CrossRef][ISI][Medline]
- Watnick,R.S., Cheng,Y.N., Rangarajan,A., Ince,T.A. and Weinberg,R.A. (2003) Ras modulates Myc activity to repress thrombospondin-1 expression and increase tumor angiogenesis. Cancer Cell, 3, 219231.[ISI][Medline]
- Vu,T.H., Shipley,J.M., Bergers,G., Berger,J.E., Helms,J.A., Hanahan,D., Shapiro,S.D., Senior,R.M. and Werb,Z. (1998) MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell, 93, 411422.[ISI][Medline]
Received November 6, 2003;
revised February 5, 2004;
accepted February 20, 2004.