Key words breast cancer - RAS - renin - angiotensin - angiogenesis - VEGF
Schlüsselwörter Mammakarzinom - RAS - Renin - Angiogenese - VEGF
Introduction
Growth and metastasis of malign tumors depends on angiogenesis in order to link the
growing cancer tissue to blood supply. The safekeeping of nourishment is thereby controlled
by self-regulated gene expression of angiogenic genes in those cancer cells causing
tumorangiogenesis. Therefore, this capacity of inducing angiogenesis has great importance
for proliferation, invasion and metastasis [1 ], [2 ]. It has been shown that tumorangiogenesis occurs differently in cancer tissue such
as breast cancer [3 ]. This finding is caused by increased expression of proangiogenic factors in cancer
cells, which lead to an imbalance of pro- and anti-angiogenic factors. One of the
most important factors regulating angiogenesis is vascular endothelial growth factor
(VEGF), which induces and controls proliferation and differentiation of endothelial
cells, tube formation and vascular maturation [4 ], [5 ]. VEGF is overexpressed in most tumors [6 ], [7 ]. Thus, in the meantime targeting VEGF by VEGF-antibodies or VEGF-traps is a well
established therapeutic strategy in clinical daily routine [8 ], [9 ], [10 ]. Expression of VEGF itself is regulated by several different upstream pro- and anti-angiogenic
factors and systems [11 ]. One of those systems is the renin-angiotensin-system (RAS), which is responsible
for regulation of renal homeostasis and the vascular tone in the cardiovascular system
[12 ], [13 ].
Angiotensinogen (AGT) becomes converted via katalytic activity of renin to angiotensin
I, and angiotensin I via angiotensin-converting enzyme (ACE) to angiotensin II, which
is the main effector of the RAS. It mediates its effects by binding to four different
angiotensin II-receptors. The most important ones are angiotensin II type 1 receptor
(AT1 R) and angiotensin II type 2 receptor (AT2 R) [14 ]. Both are g-protein-coupled receptors, whereas the activating effects are mainly
mediated via AT1 R and antagonised via AT2 R [15 ], [16 ], [17 ], [18 ]. Besides the above mentioned pathway of angiotensin II synthesis, an alternative
way has been described via angiotensin 1-12, which is expressed independently from
renin and which becomes also converted to angiotensin II by enzymatic activity of
ACE and chymase mainly in the local tissue [19 ], [20 ], [21 ]. In addition, there is a further cascade with antagonistic activity in the RAS,
since angiotensin-converting enzyme type 2 (ACE 2) converts angiotensin I into angiotensin
1-9, which on this part becomes converted to angiotensin 1-7 via ACE [22 ] ([Fig. 1 ]). ACE 2 is also able to mediate the conversion from angiotensin II to angiotensin
1-7. Angiotensin 1-7 activates the mas-receptor [23 ], [24 ] and thereby mediates mainly antagonistic effects as compared to angiotensin II.
This effects result in vasodilatation and anti-angiogenic activity [25 ]. Moreover, both players antagonise each other, since angiotensin II inhibits ACE
2- and angiotensin 1-7 increases ACE 2-expression [26 ] ([Fig. 1 ]). Finally, the local RAS contains of two different axes of angiotensin, and it has
to be assumed that influencing the systemic RAS via ACE-inhibitors and AT1 R-inibitors might also affect the balance of local RAS with regard to potential anti-angiogenic
therapeutic strategies.
Fig. 1 Cascade of major components of the RAS and their receptors.
Therefore, we addressed the question if there is a role of the RAS in the regulation
of angiogenesis in hormone-receptor positive (HR-positive) and hormone-receptor negative
(HR-negative) breast cancer cells. We investigated if angiotensin II synthesized locally
by cancer cells increases the VEGF-expression and if the VEGF-expression can be influenced
by inhibition of ACE, AT1 R and AT2 R. In addition, suppression of the RAS was performed by knockdown of AGT in order
to analyse a potential effect on VEGF with possible differences in the regulation
of tumorangiogenesis between HR-positive and HR-negative breast cancer cells with
regard to future therapeutic anti-angiogenic strategies in breast cancer.
Material and Methods
Cell cultures for breast cancer cell lines
Overall, 6 different cell lines were cultivated: 3 hormone-receptor positive (MCF-7,
ZZR-75-1, MDA-MB 361) and 3 hormone-receptor negative cell lines (MDA-MB 231, MDA-MB
468, MDA-MB 453). MCF-7, ZZR-75-1, MDA-MB 361 were cultivated in RPMI 1640 (PAA Laboratories,
Pasching, Austria), MDA-MB 231 und MDA-MB 468 in DMEM with High Glucose (4,5 g/l)
and MDA-MB 453 in DMEM/Hamʼs F-12 (PAA Laboratories, Pasching, Österreich) supplemented
by 1% Penicillin/Streptomycin (PAA Laboratories, Pasching, saturated humidity and
an atmosphere containing 5% CO2 . Media were changed every 48 h.
Stimulation with angiotensin II
Cultivated breast cancer cells were stimulated with 10−7 mol/l angiotensin II. For differentiation between the effects of extrinsic and intrinsic
angiotensin II, incubation with 10−6 mol/l ACE-inhibitor captopril was performed in order to inhibit the expression of
endogenous angiotensin II. This experiment was done with and without simultaneous
stimulation of extrinsic angiotensin II. Furthermore, in order to investigate across
which angiotensin II-receptor mediates a possible increased VEGF-expression, both
receptors have been blocked using specific inhibitors (AT1 R: 10−6 mol/l candesartan; AT2 R: 10−6 mol/l PD 123,319). This inhibition was again performed with and without simultaneous
treatment of extrinsic angiotensin II.
AGT-knockdown
In order to exclude the influence of intrinsic angiotensin II on the expression of
VEGF of the breast cancer cells, a knockdown of AGT was performed by transfection
with siRNA:
8 × 105 cells were seeded into primaria cell culture flasks T25 (BD Falcon) and cultured
under normal growth conditions (37 °C; 5% CO2 ). After 24 hours, a transfection reagent was added containing 400 µL of culture medium
without serum, 6 µL of small interfering RNA (siRNA) of negative control (Qiagen 1 027 281
20 nmol, Qiagen) or a mixture of AGT-1-siRNA, AGT-4-siRNA and AGT-7-siRNA (Qiagen,
Hildesheim, Germany). The transfection medium was incubated at least for 5 minutes
to allow the formation of transfection complexes. Cells were seeded under their normal
growth conditions with the growth medium and the transfection complexes. After 48
hours and 96 hours transfection was repeated. 48, 72, 96, 120 and 144 hours after
the initial transfection, cells with culture medium were removed and the RNA was isolated
for confirmation of a successful knockdown.
RNA Isolation and reverse transcription
Total RNA from the cultivated cells was extracted using the RNeasy mini kit (Qiagen,
Hilden, Germany) according to the manufacturerʼs instructions. The amount of RNA was
quantied by absorbance at 260 nm (DU640, Beckmann, USA) and 2,5 g of total RNA was
reverse transcribed into cDNA using random hexamer primers according to the manufacturerʼs
instructions (Applied Biosystems, Foster City, USA).
RT-PCR
RT-PCR was used for the detection of the housekeeping gene (GAPDH), as well as AT1 R, AT2 R, AGT, ACE, and VEGF expression. Four microliters of a 1 : 10 dilution of the transcribed
cDNA was used as tem-plate. PCR amplification was carried out using dNTP, forward
primer, reverse primer, MgCl2, Taq polymerase (Quiagen, Hilden,Germany) in the recommended
buffer. Amplification involved 45 cycles in an Eppendorf Thermocycler. The total volume
of the PCR reaction was 50 µl. The primers were designed according to the known sequences
of GAPDH in order to amplify a DNA of 657 bp (forward primer: 5-CTG GCGCTG AGT ACG TCG-3;
reverse primer: 5-TTG ACAAAG TGG TCG TTG A-3), AT1 R in order to amplify a DNA fragment of 330 bp (forward primer: 5-GGA AAC AGC TTGGTG GTG AT-3;
reverse primer: 5-GCA GCC AAATGA TGA TGC AG-3), AT2 R in order to amplify a DNA fragment of 263 bp (forward primer: 5-CTG CTG TTGTTC TGG CCT TCA T-3;
reverse primer: 5-ACT CTCTCT TTT CCC TTG GAG CC-3), AGT in order to amplify a DNA
fragment of 499 bp (forward primer: 5-CCC TGG CTT TCA ACACCT AC-3; reverse primer:
5-CTG TGG GCT CTC TCTCAT CC-3), ACE in order to amplify a DNA fragment of 428 bp (forward
primer: 5-GGT GGT GTG GAA CGAGTA TG-3; reverse primer: 5-TCG GGT AAA ACTGGA GA TG-3),
and VEGF in order to amplify a DNA fragment of 367 base pairs (bp) (forward primer:
5-CGG GCC TCC GAA ACCATG AAC TTT-3; reverse primer: 5-CTA TGT GCTGGC CCT GGC CCT GGT GAG GTT-T-3)
and Bands were visualised after electrophoresis on a 2% agarose gel (Invitrogen GmbH,
Karlsruhe, Germany).
Quantitative Real-Time PCR
For quantification of VEGF, AGT and β2 -Microglobulin, corresponding kits from Applied Biosystems were used according to
the instructions of the manufacturer (VEGF: Hs00 173 626_m1; AGT: Hs01 586 213_m1:
β2 -Microglobulin: 4 326 319E). The quantity of cDNA for the genes of interest was normalised
to the quantity of 18S RNA in each sample (delta-CT-method). Gene expression in the
figures is presented as 1/delta CT.
Verification of the successful AGT-knockdown
In order to prove the successful knockdown of AGT, we analysed the concentration of
AGT-mRNA using quantitative real-Time PCR. Here, indeed we did not observe a complete
switch-off of AGT, however it was possible to show a significant reduction of the
expression of AGT (p = 0.034).
Analysis of Quantitative Real-Time PCR-rawdata
The quantity of cDNA for the genes of interest was normalised to the quantity of 18S
RNA in each sample by dividing the fluorescence values for the gene amplification
with the fluorescence values for the 18S RNA amplification. Since these delta ct values
are negatively correlated with the amount of gene expression, they were converted
to 1/delta ct in order to avoid confusion.
Statistics
Statistical analysis was performed using IBM SPSS Statistics Version 21. After verification
of a normal distribution of the data received after stimulation with angiotensin II,
analysis of variance was used. In case of significant data, paired comparison after
Bonferroni was performed. Due to the fact, that the knockdown data showed no normal
distribution, statistics was calculated according the Mann-Whitney-test. Presentation
of the data is carried out using Box-Whisper-plots. Differences were considered to
be significant at p < 0.05 and significant differences between treatment arms are
marked with asterisks (*).
Results
VEGF-expression in human breast cancer cells
Experiments with human breast cancer cell lines confirmed the presence of the different
components of the RAS. In both, in the HR-positive cell lines MCF-7, MDA-MB 361, and
ZR-75-1 as well in the HR-negative cell lines MDA-MB 231, MDA-MB 453 and MDA-MB 468
gene expression of AT1 R, AT2 R, AGT, ACE, and VEGF was observed ([Fig. 2 ]).
Fig. 2 a – c Expression of the different components of the RAS in the HR-positive breast cancer
cell lines MCF-7 (a ), MDA-MB 361 (b ), and ZR-75-1 (c ) showing the RT-PCR amplification of (1) GAPDH (657 bp), (2). Negative control, (3)
AT1 R (330 bp), (4) AT2 R (263 bp), (5) AGT (499 bp), (6) ACE (428 bp), and (7) VEGF (367 bp). d – f Expression of the different components of the RAS in the HR-negative breast cancer
cell lines MDA-MB 231 (d ), MDA-MB 453 (e ) and MDA-MB 468 (f ) showing the RT-PCR amplification of (1) GAPDH (657 bp), (2). Negative control, (3)
AT1 R (330 bp), (4) AT2 R (263 bp), (5) AGT (499 bp), (6) ACE (428 bp), and (7) VEGF (367 bp).
Effect of extrinsic and intrinsic angiotensin II
Since we proofed the presence of those genes in the above mentioned breast cancer
cell lines, stimulation of the cells with extrinsic angiotensin II was performed in
order to investigate the effect on VEGF expression. We revealed a significant extrinsic
angiotensin II-dependent upregulation of VEGF in all cell lines together. However,
separated analysis of HR-positive and HR-negative cells after incubation with angiotensin
II only reached borderline significance ([Fig. 3 ]). In the next step, we focused on the meaning and the functionality of intrinsic
angiotensin II with regard to the expression of VEGF. In absence of any extrinsic
angiotensin II, the intrinsic conversion to angiotensin II was inhibited by captopril
and expression of VEGF was quantified, revealing a significant decrease of VEGF in
HR-positive and HR-negative cell lines ([Fig. 4 ]). Obviously, the angiotensin II effect is mainly mediated via AT1 R, since inhibition of AT1 R using candesartan also caused a significant decrease of VEGF in all cell lines ([Fig. 4 ]). In contrast, inhibition of AT2 R by PD 123,319 did not show any significant differences neither in HR-positive, nor
in HR-negative cells.
Fig. 3 Extrinsic angiotensin II-dependent expression of VEGF: a stimulation of all cells with extrinsic angiotensin II reveals a significant increase
of VEGF-expression as compared to controls (p = 0.038; ct = cycle threshold). b separated stimulation of HR-positive and -negative cells with extrinsic angiotensin
II again shows an increase of VEGF in both groups. However, this differences are only
of borderline significance (p = 0.091; ct = cycle threshold).
Fig. 4 Effect of intrinsic angiotensin II-dependent expression of VEGF: VEGF-expression
after treatment of cells with the ACE-inhibitor captopril and AT1 R-inhibitor candesartan shows a significant decrease in HR-positive (red) (p = 0.02)
and HR-negative (blue) cell lines (p < 0.01). In contrast, inhibition of AT2 R using PD 123,319 did not show any significant effects on VEGF in HR-positive and
HR-negative cell lines (ct = cycle threshold).
Separation of extrinsic and intrinsic effects of angiotensin II
In order to separate the effects of extrinsic and intrinsic angiotensin II on VEGF,
cells were again incubated with the ACE-inhibitor captopril to prevent intrinsic angiotensin
II-expression and simultaneously incubated with extrinsic angiotensin II. In HR-negative
cell lines, we detected a significant decrease of VEGF-expression as compared to the
controls, which we could not find in HR-positive cell lines ([Fig. 5 ]). In addition, simultaneous incubation with angiotensin II and candesartan decreases
VEGF-expression significantly in both in HR-positive and HR-negative cell lines. Furthermore,
simultaneous incubation with angiotensin II and PD 123,319 again decreases VEGF-expression
significantly in both in HR-positive and HR-negative cell lines. This decrease seems
even to be stronger as the decrease of VEGF after inhibition of AT1 R ([Fig. 5 ]).
Fig. 5 Extrinsic angiotensin II-dependent VEGF-expression after: 1. inhibition of intrinsic
synthesis of angiotensin II by captopril is significantly decreased in HR-negative
cells (blue) (p < 0.05) but shows no difference in HR-positive cells (red) (p = 0.407).
2. Inhibition of AT1 R by candesartan is significantly decreased in HR-negative cells (blue) (p = 0.004)
and HR-positive cells (red) (p < 0.001). 3. Inhibition of AT2 R by PD 123,319 is also significantly decreased in HR-negative cells (blue) (p < 0.001)
and HR-positive cells (red) (p < 0.001). This decrease seems even to be stronger as
the decrease of VEGF after inhibition of AT1 R.
Quantification of VEGF
In order to analyse the overall effect of the RAS on VEGF and angiogenesis in HR-positive
and HR-negative breast cancer cells, the last step was quantifying VEGF-expression
after having performed a knockdown of AGT in those cells. In doing so, HR-positive
cells showed a highly significantly increased expression of VEGF at any time of incubation
(48, 72, 96, 120, and 144 hours) whereas HR-negative cells only had a significant
VEGF-increase after 48 hours of incubation. Surprisingly, after 144 hours a significant
decrease of VEGF-expression could be detected ([Fig. 6 ]).
Fig. 6 VEGF-expression after knockdown of AGT via siRNA-transfection: a HR-positive cells show a highly significantly increased expression of VEGF (red)
at any time of incubation (48, 72, 96, 120, and 144 hours) (p < 0.002) as compared
to controls (red lines). ct = cycle threshold. b HR-negative cells only show significantly increased expression of VEGF after 48 hours
of incubation (p = 0.003). After 72, 96, and 120 hours no significant differences
could be observed. However, after 144 hours a significant decrease of VEGF-expression
has been detected (p = 0.04).
Discussion
Aim of this study was to investigate the RAS-dependent regulation of tumorangiogenesis
as a function of the hormone-receptor-status of breast cancer cells. The important
influence of the RAS on VEGF is well known and has been described for many different
tumor entities [27 ]. However, currently little is known concerning differences in RAS-dependent VEGF-expression
in HR-positive an HR-negative breast cancer. In this study, it has been shown that
expression of VEGF was increased due to extrinsic as well as intrinsic angiotensin
II in all investigated breast cancer cell lines. This stimulating effect is mediated
via the AT1 R whereas the AT2 R has a more modulating function. In addition, it was shown that knockdown of AGT
increases VEGF significantly in HR-positive breast cancer cells but decreases VEGF
in HR-negative cells. This indicates that RAS-dependent tumorangiogenesis in HR-positive
and HR-negative breast cancer cells is regulated differently.
For many type of tumors, an important role of the RAS has been shown including cancers
of the prostate, brain, cervix, pancreas und lung [28 ]. In particular, this concerns presence of AT1 R, which is necessary for mediation of the proangiogenic effects of angiotensin II
[29 ], [30 ]. Here, we showed the expression of AGT, ACE, AT1 R, AT2 R, and VEGF in all analysed cell lines, fulfilling the requirement for the hypothesis
that tumorangiogenesis can be regulated by the RAS basically in HR-positive and HR-negative
breast cancer cells. This is in line with immunohistochemical data of Jethon et al
[31 ], who detected the AT1 R in both HR-positive and HR-negative breast cancer tissue. Former data showed, that
angiotensin II has to be considered as the main player for proliferation of tumor
cells as well as endothelial cell, thus angiogenesis mediating this effect via AT1 R [32 ], [33 ]. Therefore, it can be hypothesized that the RAS is able to regulate VEGF and thereby
influences angiogenesis in breast cancer. Due to the worse prognosis of HR-negative
as compared to HR-positive breast cancer, it might have been assumed that VEGF-expression
differs between those tumor types, however this could not be confirmed. Epidemiological
studies showed, that women with decreased angiotensin II levels due to defect enzymes
have a reduced risk for breast cancer [34 ], [35 ]. In summary, obviously the extrinsic stimulation of angiotensin II can not explain
the different regulation of angiogenesis between the different breast cancer types,
but can be used in order to describe the function and interaction of the different
receptors.
In many tumors, the AT1 R is overexpressed [29 ] and angiotensin II-dependent upregulation of VEGF is mediated mainly via AT1 R [33 ]. In contrast, there is only rare and inconsistent data concerning the meaning of
the AT2 R for tumorangiogenesis. Concerning VEGF there is data for agonistic as well as antagonistic
effects mediated by AT2 R [36 ], [37 ], [38 ], [39 ]. According to the published literature, at first glance parts of our data seem also
to be conflicting, since on the one hand we showed that inhibition of the AT2 R with PD 123,391 and simultaneous stimulation with extrinsic angiotensin II decreased
VEGF. On the other hand, the sole inhibition of AT2 R did not influence the amount of VEGF. Obviously, the AT2 R does not act exclusively antagonistically, but has a more modulating effect in case
of simultaneous activation of AT1 R. Therefore, it seems that angiotensin II can perform its increasing effect on VEGF
only after co-activation of AT2 R. This hypothesis is also supported by Clere et al., who showed that AT2 R-mediated effects differ depending on the type of cells and the physiological context
[40 ].
As discussed above, an extrinsic angiotensin II-dependent pathway controlling tumorangiogenesis
in breast cancer is rather unlikely. It has much more to be assumed that the intrinsic
angiotensin II, which is expressed by cancer cells themselves, is more important.
Here, an autocrine stimulation of cancer cells followed by upregulation of VEGF seems
possible. Incubation of the breast cancer cell lines with the ACE-inhibitor captopril
prevented the synthesis of intrinsic angiotensin II, which might initiate an autocrine
stimulation of VEGF-expression. Accordingly, a significant decrease of VEGF was detected.
This result is in line with Koh et al. [34 ] und Gonzalez-Zuloeta et al. [35 ], who showed that genetically altered activity of ACE is associated with increased
or decreased risk for breast cancer. However, there was no difference between the
risk for HR-positive and HR-negative breast cancer. In contrast, only in HR-positive
breast cancer cells, this decrease of VEGF after having prevented the production of
intrinsic angiotensin II can be avoided by treatment with extrinsic angiotensin II.
Besides ACE, there are a couple of further enzymes such as chymase, catalysing the
conversion from angiotensin I to angiotensin II [41 ], [42 ]. Therefore it has to be assumed that a possible therapeutic anti-angiogenic strategy
via the RAS should focus on inhibition of the AT1 R instead on ACE, but up to date, concerning this consideration, there is no data
available. Treatment with candesartan still allows production of intrinsic angiotensin
II, but obviously the level of VEGF is decreased due to the prevented autocrine stimulation.
Consistent with our data in breast cancer cells, this has been presented for other
tumor entities such as ovary [43 ], prostate [44 ] and pancreas [45 ]. However, inhibition of the AT2 R does not increase VEGF, which allows to hypothesize that in contrast to others [46 ] inhibition of the AT2 R does not antagonize the effect of AT1 R-mediated angiogenesis directly.
Despite many published studies concerning the RAS and its role for tumorangiogenesis,
the exact mechanism remains still unclear. In order to evaluate the total effect of
the RAS on tumorangiogenesis in breast cancer, we performed a knockdown of AGT and
revealed different results for HR-positive and HR-negative breast cancer cells. Although
a complete knockoff of AGT was not achieved, we detected for the first time a significant
upregulation of VEGF in HR-positive cells but a significant decrease of VEGF in HR-negative
cells after 144 hours of incubation. Obviously, although we achieved only a partial
knockdown of AGT, this knockdown was still strong enough in order to influence VEGF
as described. These results can be explained by looking at a further member of the
RAS, angiotensin 1-7. In the mouse model as well as in lung cancer cells, angiotensin
1-7 reduced growth of cancer cells and/or inhibited angiogenesis [47 ], [48 ]. This anti-proliferating and anti-angiogenic effect is thereby mediated by the suppression
of VEGF [46 ], [48 ].
In summary, the percentage of the multiple RAS-mediated effects differs between HR-positive
and HR-negative breast cancer cells. It seems possible that in HR-positive cells,
the RAS acts more anti-angiogenic by influencing the angiotensin 1-7/mas-pathway,
antagonising a high intrinsic VEGF-expression. In contrast, in HR-negative cells the
focus of the RAS-effects is more angiotensin II/AT1 R-based and therefore pro-angiogenic via increased intrinsic VEGF. However, further
data is needed in order to estimate, if influencing the RAS might be a future anti-angiogenic
acting component in the multi-modal therapy of breast cancer patients.
Conclusion
RAS-dependent regulation of VEGF between HR-positive and HR-negative human breast
cancer cells seems do be different. These findings provide evidence for a possible
future therapeutic strategy.
