Carcinogenesis Advance Access published March 4, 2004

Carcinogenesis 2004 # Oxford University Press; all rights reserved.

Akt mediates an angiogenic switch in transformed

keratinocytes

Carmen Segrelles1, Sergio Ruiz 1, Mirentxu Santos 1, Jesús Martínez-Palacio1, M.

Fernanda Lara and Jesús M. Paramio1,2

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1.- Department of Cell and Molecular Biology, CIEMAT, Av. Complutense 22, E-28040
Madrid, SPAIN.
2.-To whom correspondence should be addressed:
Department of Cell and Molecular Biology and Gene Therapy,
CIEMAT (ed. 7),
Av. Complutense 22,
E-28040 Madrid (SPAIN).
Phone 34+ 91346 6051
Fax: 34+ 91346 6484
e-mail: jesusm.paramio@ciemat.es
 ABSTRACT

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
VEGF protein levels. However, in contrast to v-rasH a-transformed keratinocytes, VEGF
mRNA levels were not increased. The induction of VEGF protein by Akt is associated

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with increased phosphorylation and thus activation of p70S6K and 4E-BP1, leading to
increased VEGF translation. In addition, we observed increased MMP2 and MMP9
expression, but not TSP1, 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.

INTRODUCTION
The mouse skin carcinogenesis model has provided an important instrumental
framework for understanding many of the concepts currently applied human neoplasia
(reviewed in [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,
overexpression 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 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 K10 - which 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 [11-13].
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 [18-24].

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 There is evidence that suggests the PI3K/PTEN/Akt pathway may be involved in
tumor angiogenesis (reviewed in [25]). This appears to proceed mainly through the
regulation of VEGF expression [26-28] 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 inc reased p70S6K and
4E-BP1 phosphorylation. Further, Akt-induced tumors also show increased MMP2 and

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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.

MATERIALS AND METHODS

Cell culture, transfection and in vivo tumorigenic assays. Mouse PB
keratinocytes [30] and Akt-transfected derivatives (as 40-80 pooled clones) were grown
and subcutaneously injected into nu/nu mice as previously reported [3]. In some cases,
prior to subcutaneous injection, the cells were either mock- or retrovirus-infected as
previously described [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 two days [volume was calculated as ? (4/3) x
(width/2) 2 x (length/2)]. One hour prior sacrifice, mice were subcutaneously injected with
BrdU (0,1mg/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) 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 (Quiagen)
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). 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 and used at 15mM, 10nM and 1nM respectively. These drugs were added to the
transfected cells 24 hours after transfection and the cultures were incubated for further
24 hours. 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) and sectioned (6?m
thick) were fixed in acetone at -20ºC for 5 min and the blood vessels visualized using rat
anti-mouse CD31 (Pharmingen, diluted 1:30). To monitor vessel maturation, sections

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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 were determined (using Microimage 4 software - Olympus) 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 4-6 individual tumor samples.

Northern blotting. Total RNA from different tumors or from pooled cultured
clones (20-40 different clones each) was isolated by the guanidine isothyocianate-

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phenol-chloroform extraction. and probed by Northern blotting (15 ? g/lane) for VEGF
expression using mouse-specific VEGF probes as previously described [15]. Equal
loading was confirmed by hybridization with a 7S RNA probe. Quantification was
performed using a Phosphorimager and Quantity One software (Bio Rad)

Immunohistochemistry. The detection of CD·31, ? -actin, MMP-2, MMP-9,

VEGF and TSP-1 was performed in frozen sections in parallel with antibodies reacting
with mouse keratins (either rabbit polyclonal against K5 or mous monoclonal against
K14 essentially as previously described [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 4 tumors of each type were analyzed. Controls omitting
primary antibodies, or after the preincubation 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 (Sta. Cruz Biotech.), HIF1? [35,36] (Transduction Labs),
phosphorylated Akt (Ser473; Cell Signaling), phosphorylated p70S6Kinase (Thr389 [37];
Thr421/Thr424 [38]; Cell Signaling), phosphorylated 4E-BP1 (Ser65; Cell Signaling [39]),
MMP-2 [40], MMP-9 [41], VEGF [42] and TSP-1 [43] (Neomarkers). 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)

RESULTS AND DISCUSSION

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, is poorly tumorigenic in xenograft
experiments, displaying long latency, reduced growth rate and the tumors obtained show
highly differentiated phenotypes (Fig. 1A, B, C and D; 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 (Fig. 1A, B, C', D; see also [3]). Analysis of these tumors clearly
demonstrated that Akt expression leads to increased proliferation and reduced apoptosis

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(Fig 1E) in agreement with our previous data [3]. However, the increased proportion of
cells in S phase upon Akt transfection in vitro (Fig 1F) does not seem to account for the
growth rate observed in the tumors in vivo (Fig 1B) and suggests the existence of other
mechanisms that support tumor development.
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 upregulation 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

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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 (Fig 2A).
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
(Fig. 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 (Fig 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 (Fig 2 C, 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 to
control samples (Fig. 3A, B). On the other hand, the mean size of the vessels was larger
in control than in Akt-induced tumors (Fig. 3C). Collectively, these data show that Akt
induces an angiogenic switch in keratinocytes.

Akt-induced angiogenesis is not mediated by increased VEGF mRNA

VEGF mediates the essential angiogenic response required for mouse skin
tumor progression [15-17]. 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 tumor-
stroma ratio were used in Northern blot analysis. No significant differences were seen
among the different samples (Fig. 4A, 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 (Fig. 4B, 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-ras H a expression. Empty vector and Akt-
transfected PB keratinocytes were transduced with a v-rasH a-coding retrovirus. The
mock or v-ras H a 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-ras H a, Akt and Akt plus v-ras Ha). The obtained results (Fig. 4C, C’)
confirmed the induction of VEGF mRNA expression mediated by ras, in agreement with

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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 (Fig. 4C). Collectively, these data
indicated that Akt is not involved in VEGF mRNA expression in PB keratinocytes.
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 (Fig 4D). The present observations

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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 upregulation, the observation that Ha ras induces VEGF
mRNA in parental or Akt-transfected keratinocytes (Fig. 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 (Fig 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 [55-58]. 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 (Fig. 5A, A’). In
addition, the expression of VEGF protein in tumors derived from Akt-transfected
keratinocytes was increased (Fig. 5A), while no increase in HIF1? ?protein expression
was seen (Fig. 5A). Given that the transcriptional activation of VEGF is mainly mediated
by this transcription factor, the absence of increased HIF1? ?protein levels might
correspond to the lack of induction of VEGF mRNA observed (Fig. 4). The transcriptional
activation of VEGF is mainly mediated by the transcription factor hypoxia-inducible factor
1 (HIF-1? ). The present data, which show no inc rease in HIF-1? ?protein (Fig. 5A, A’), are
therefore in agreement with the lack of change in VEGF mRNA in cells and tumors (Fig.
4).
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 [56,57,60-62]. 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'

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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 eIF4E-binding
proteins (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 coordinately 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 kinasewas 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

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keratinocytes compared to those derived from control keratinocytes (Fig. 5B, 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 (Fig. 5B, B’).
At present we may not discern, whether eIF4 or p70S6K-dependent mechanism
is the 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 [65,70-74], thus suggesting the involvement of mTOR in the increased VEGF
protein translation. In agreement, preliminary experiments indicate that rapamycin
treatment decreases tumorigenic behaviour of Akt and Ha-ras transduced keratinocytes
(Segrelles et al., unpublished results). Although the potential involvement of
antiangiogenic process has not been yet tested, these data indicates that mTOR acts
downstream of Akt and Ha-ras in keratinocyte transformation. Collectively, these data
indicate that 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, HIF-1?
expression has also been described to be under the control of the
PI3K/PTEN/Akt/mTOR pathway [75-77]. Given the increased Akt activity in the tumors
(Fig. 5, see also [3,44]), the lack of increased levels of HIF-1? 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 [19,20,80-82], whereas thrombospondin-1 (TSP-1) is
considered an inhibitor [20-23,83]. 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 (Fig. 5C, C’) in agreement with
previous data [18,84-86]. On the other hand we found that TSP-1 levels were similar in
the different samples (Fig. 5C , 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

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recently demonstrated that the activation of Rho mediated by increased PI3K activity is
responsible for the downregulation 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 (Fig. 5), Akt led to increased VEGF, MMP2 and MMP9
expression (Fig. 6A', B’ C') compared to control samples (Fig. 6A, B C). Interestingly, the
expression of MMP2 and MMP9 was found mainly in the tumor periphery and few tumor
cells, either Akt or v-ras H a (Fig 6 B’,B’’, C’, C’’, respectively) also display

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immunoreactivity against these molecules, thus suggesting that tumor cells are not the
primary source of these metaloproteinases. On the other hand, no changes in TSP1
expression was observed between control and Akt-derived tumors (Fig. 5D, D'),
whereas v-ras H a transduction led to decreased expression (Fig. 5 D'').
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 than the may secrete other molecules such 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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 FIGURE LEGENDS
Fig. 1 Increased tumorigenic potential of Akt transfected keratinocytes. Pooled
clones (20-40) 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, D) Histological appearance of tumors from control keratinocytes showing well-
differentiated morphology (C), whereas Akt transfection leads to poorly differentiated
(C', 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

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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 ? S.D. F) Percentage of Akt- and pcDNA-transfected pooled clones in S-phase
in vitro analyzed by FACS after being stained with propidium iodide.

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, 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, 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, C’) was used to counterstain tumoral cells. Arrows in B denote
lacunar blood vessels commonly observed in control tumors. Bars = 100 ? m.

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 to control samples. At least 5
tumors of each type were analyzed using 4-6 fields on each. Data are shown as
mean ? S.D.

Fig. 4. Akt does not induce VEGF mRNA. Total mRNA from different tumor samples
(A, 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 probed was used
in all the cases . Note that no increase in VEGF expression was observed in Akt-
derived samples, whereas v-ras Ha infection promoted the increased VEGF mRNA
expression both in control and Akt-derived tumors (C). A’, B’ C’) Semiquantitative
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 hours with the stated
inhibitors. Data in D, D’ come from three independent experiments and are shown as
mean ? S.D.

Fig. 5. Akt induces VEGF, MMP2 and MMP9 proteins. A) Protein extracts from
different tumors obtained with Akt-transfected cells and from control cellss (including
well-differentiated (w-d) and poorly differentiated (p-d)) were probed by Western

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 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’, B’ C’) Semiquantitative analysis
obtained by densitometry of the corresponding western blot data.

Fig. 6. Immunohistochemical detection of VEGF, MMP2 and TSP1 in tumors.

Frozen sections of tumors obtained after subcutaneous injection of parental (A; B; C,

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D), Akt-transfected (A’, B', C', D’), and control keratinocytes upon v-rasH a infection
(A’’, B’’, C’’, D’’), were stained for VEGF (green in A, A’, A’’), MMP2 (green in B, B',
B'’),MMP9 (green in C, C', C'') and TSP-1 (green in D, D', D'’). Together with K14 or
K5 (in red) to denote the tumoral cells. Note that the Akt and v-ras Ha infection led to
increased VEGF, MMP2 and MMP9 protein expression, whereas only v-rasH a
infection decreased TSP1 expression. In addition, MMP2 and MMP9
immunoreactivity is predominant at the tumor boundary (B’,B’’, C’, C’’) Bar= 50? m.

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 ACKNOWLEDGEMENTS
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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