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. Author manuscript; available in PMC: 2018 Dec 31.
Published in final edited form as: J Exp Ther Oncol. 2017 Sep;11(2):107–115.

Angiotensin receptor signaling and prostate tumor growth in mice

Jem Scott-Emuakpor 1,9, Emma Allot 3,5,6, Stacy A Johnson 1,2,4,7, Lauren E Howard 8, Everardo Macias 5, Stephen J Freedland 1,3,5,6, Susan B Gurley 1,2,4,7
PMCID: PMC6311706  NIHMSID: NIHMS999417  PMID: 28976133

Abstract

The renin-angiotensin system, through its type 1 and type 2 angiotensin receptors (AT1R and AT2R, respectively) may have a role in prostate cancer. The objective of this pilot study was to explore that potential role by determining whether the AT1R blocker, losartan, would reduce the growth of LAPC-4 prostate cancer xenografts in nude mice. We also evaluated the tumor growth effects of using angiotensin II to activate both AT1R and AT2R simultaneously. Our data showed that losartan decreased tumor volumes by 56% versus control. This decrease reached statistical significance at day 54 (p = 0.0014). By day 54, Ki67 was also reduced in the losartan group, though not significantly so (p = 0.077). Losartan had no significant effect on AT1R or AT2R expression. Despite significant increases in both AT1R and AT2R at day 29 (p = 0.043 and 0.038, respectively), the administration of angiotensin II did not result in any significant differences in tumor volumes or ki67 at any time point. These data suggest that selective activation and induction of AT2R coupled with blockade of AT1R may slow prostate cancer growth. Future larger studies are needed to confirm these results.

Keywords: AT1R, AT2R, mouse, RAS, angiotensin, tumor, cancer, prostate

INTRODUCTION

Prostate Cancer (PC) is the second leading cause of cancer death in men in the United States and accounts for 26% of newly diagnosed cancers in men (1), yet its pathogenesis is not completely understood. Available systemic therapies for PC are often initially efficacious, but patients may become resistant (2). Thus, better therapies are needed.

Previous studies indicated that the renin-angiotensin system (RAS), which plays a major role in cardiovascular function, may be important in the development of PC (2–4). These studies found the hormone, angiotensin II, in multiple PC lines, while in normal prostate, angiotensin II is only found in the basement membrane (4). Angiotensin II works via several receptors, including type 1 (AT1R) and type 2 (AT2R). AT1R mediates many of the cardiovascular effects of the RAS and is the target of commonly-prescribed angiotensin receptor blockers (ARBs), such as losartan. AT2R, normally expressed at low levels (5), may have a protective role in pathologic conditions, as demonstrated by rodent models of myocardial infarction (6), pancreatitis (7), and chronic kidney disease (8).

Through AT1R, angiotensin II plays a significant part in promoting mechanisms related to prostate tumorigenesis and metastasis, including angiogenesis, cell proliferation, and tissue remodeling (9–11). Despite the implication that AT1R blockade may inhibit tumorigenesis, most observational studies noted either an increased overall risk for cancer (12–14) or no effect at all (15) in patients using ARBs, which selectively block AT1R without reducing the availability of angiotensin II.

Studies investigating the role of AT2R in PC suggest that increased expression of AT2R has an inhibitory effect on various adenocarcinomas by facilitating anti-neoplastic processes, such as apoptosis and cell differentiation (10,16,17). One study found that cancers with higher Gleason scores expressed less AT2R compared to cancers with lower Gleason scores (18).

Based on available data, the exact role of the RAS in PC remains unclear. However, given the potentially tumorigenic effects of angiotensin II, we hypothesized that using losartan to inhibit AT1R signaling would reduce PC xenograft growth in mice. We also hypothesized that angiotensin II, which activates both AT1R (pro-tumor) and AT2R (anti-tumor) with their opposing effects on tumor growth and progression, would result in no difference in tumor growth relative to untreated controls.

MATERIALS AND METHODS

Animals, husbandry, and cell culture

Animals

Thirty Male Balb/c nude mice, at 6–8 weeks of age, were received from Taconic Farms, Germantown, NY and acclimated for 7 days before study initiation. Animals were housed in the Durham VA Medical Center’s AAALAC-accredited Animal Research Facility and observed for mortality/moribundity twice daily.

Xenografts

For this study, we chose LAPC-4, because it is an androgen-sensitive prostate cancer cell line that maintains a wild-type androgen receptor (as opposed to LnCaP) and does not express androgen-receptor variants (as opposed to CWR22rv1). As such, we believed this was a reasonable model for early stage, androgen sensitive prostate cancer.

LAPC-4 human PC cells, donated by Dr. William Aronson of University of California, Los Angeles (Los Angeles, CA), were prepared as previously described (19). On Day 21 of study (7 days after arrival), mice were weighed then injected subcutaneously with 1 × 106 LAPC-4 prostate tumor cells suspended in a 100 μl of a 1:1 mixture of media and Matrigel® (Becton Dickinson, Franklin Lakes, NJ). Cells were injected into the right, rear flank of each mouse. Once palpable, tumor dimensions were measured weekly, using digital calipers. Tumor volumes were calculated using the formula (width2 × length)/2.

Twenty-one days after tumor implantation, mice were randomized by tumor size to ensure equal tumor sizes across three treatment groups (n = 10/group): untreated control, losartan, or angiotensin II. See Figure 1 for study timeline.

Figure 1.

Figure 1.

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All animals were acclimated for 7 days prior to prostate cancer cell implantation. Dosing began 3 weeks after tumor cell implantation and was discontinued at 29 days (50 days after implantation). SAC = scheduled sacrifice; Ang II = angiotensin II.

Treatment groups

On the first day of treatment, the angiotensin II group underwent aseptic surgery to subcutaneously implant osmotic minipumps (DURECT Corporation, Cupertino, CA). Each minipump administered angiotensin II at a sub-hypertensive dose of 10.8 ng/min.

The losartan group received losartan, administered via drinking water, at a concentration of 66.7 mg/L. Based upon a presumed water intake of 4.05 ml/mouse/day (20), this amounted to 0.27 mg of losartan per mouse per day. As mice were group housed and water intake was not measured, the exact dose of losartan for each mouse is unknown.

Outcomes

Each animal was weighed prior to treatment start and at necropsy, after the removal of the osmotic minipump and excision of the tumor.

Treatment with losartan or angiotensin II was discontinued at 29 days in all mice. At that time (50 days after tumor implantation), half of the mice were humanely euthanized, while the remaining were euthanized 76 days after implantation.

The limited necropsy included gross examination of organs. Excised tumors were immediately weighed. A representative section was collected in 10% neutral buffered formalin and the rest was flash frozen in liquid nitrogen then stored at −80°C.

Histopathology

Formalin-fixed tumors were embedded in paraffin, and slices were mounted onto slides for H&E staining. Morphological and mitotic analyses were performed by Antech Diagnostics (Irvine, CA). Mitotic activity was quantified as the number of mitotic bodies per 10 high-power fields.

Ki67 analysis was performed by the Diagnostic Center for Population and Animal Health, Michigan State University (East Lansing, MI) as previously described (21). Aside from one sample, which, because of its small size, had only four fields counted, five consecutive fields were counted for each.

RNA isolation and analysis

Total RNA was extracted from the frozen tumor samples using the RNeasy kit (Qiagen, Germantown, MD) according to manufacturer instructions. cDNA was generated by reverse transcription, using the qScript cDNA super mix (Quanta BioSciences, Gaithersburg, MD). The mRNA levels for AT1R and AT2R were measured using quantitative, real-time PCR. Quantitative PCR was performed using the SYBR Green method, based on previously published sequences (22). Gene expression was quantified using the ddCt method for quantitation (23) relative to the control group and normalized to GAPDH expression.

Statistics

Results are represented as mean ± SEM. Final tumor weights and gene analysis data were compared between the experimental groups and control using two-tailed student’s t-test. Tumor growth volumes were compared between the experimental groups and control using a longitudinal analysis. The longitudinal analysis was conducted using a generalized linear model with treatment effect, time effect, and treatment by time effect to test whether there was a difference in tumor volume across different treatment groups over the 50 days post randomization (See Figure 2A). Time was treated as categorical since the growth patterns in each treatment group were not linear.

Figure 2.

Figure 2.

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(A) Weekly tumor volumes (mm3) showing growth trends over 50 days of study, beginning with the first day of treatment (the day animals were randomized into treatment groups). Treatment with either losartan or angiotensin II was discontinued at 29 days, with 56% smaller tumors in the losartan group versus control (n = 9/group). This difference reached significance at 50 days (n = 5/group; p = 0.0014 vs control (longitudinal analysis)). (B) Final tumor weights (mg) showing a similar growth pattern to weekly tumor volumes (29 day: n = 4/group; 54 day: n = 5/group).

P<0.05 was regarded as statistically significant. However, as this was a pilot study, we also used p<0.10 to define findings that were of interest and would justify repeating using larger sample sizes.

RESULTS

Survival and exclusions

Twenty-seven out of 30 animals survived until their scheduled sacrifices. One animal from the control group and two from the angiotensin II group were found dead prior to Day 15 of study. All three were tumor-bearing, with tumors smaller than 150 mm3. As none showed clinical signs of illness or injury prior to being found dead, their deaths were deemed to be unrelated to treatment. Data from these three animals were removed from the final analyses.

None of the 27 surviving animals showed clinical signs attributable to tumor burden, angiotensin II administration, or losartan administration. Immediately prior to scheduled necropsies, all 27 animals appeared healthy. Body weights remained stable at each time point and there were no significant changes in group mean body weights throughout the study (data not shown).

Tumor growth volumes

At randomization (the first day of treatment), tumor volumes were similar across groups, with group averages ranging from 102.08 mm3 to 108.03 mm3 (p>0.1). At 22 days of treatment, group mean tumor volumes began to diverge, with the control group having the largest tumor volumes, followed closely by the angiotensin II group, and with the losartan group having 56% smaller tumors than the control group by 29 days.

At 29 days, half the mice in each group were euthanized and all treatments discontinued, leaving only 5 mice/group. Despite cessation of treatment, tumor growth differences persisted, with control having the largest tumors, followed by the angiotensin II group, and with the losartan group continuing to grow 56% smaller tumors than the control.

There were no differences in tumor volumes between the control group and the angiotensin II group throughout the study (p = 0.1). However, group mean tumor volume was significantly smaller in the losartan group (617.9 ± 193.3 mm3) versus control (1394.2 ± 467.9 mm3) at 50 days post-randomization (p = 0.0014). See Figure 2 for tumor growth volumes.

Post mortem findings

There were no abnormal findings on gross necropsy at either time point in any mice with the exception of two (one in the losartan group and one in the angiotensin II group) with masses that were determined, upon histology, to be chronic cellulitis.

All tumors were firm and lobulated. After weighing, tumors were transected. Tumors weighing greater than 0.5 mg contained purulent or caseous fluid, while those under 0.5 mg were solid throughout. This was true regardless of treatment arm.

Consistent with tumor volume measurements, control mice had the largest tumors at both times points, while the losartan group had the smallest. However, none of these differences were statistically significant (p>0.1). See Figure 2 for tumor volumes and final tumor weights.

Histologic findings

H&E staining

Tumors consisted of multinodular areas of neoplasia, which infiltrated the surrounding tissue. Reactive connective tissue, macrophages, plasma cells, and neutrophils could also be seen. There was no consistency in mitotic activity within groups; mitotic activity ranged from 0-24/10 hpf with no differences between groups at any time point. Each sample of neoplastic tissue was identified as adenocarcinoma.

Ki67 staining

Quantitative analysis of Ki67 staining at 29 days, revealed no significant differences among groups (148.4 ± 20.9/40 hpf; 130.2 ± 36.1/40 hpf; 120.5 ± 10.8/40 hpf, in the losartan, angiotensin II, and control groups, respectively).

At 54 days, Ki67 staining in the losartan group (109.0 ± 33.7/40 hpf) was less than that in the control group (182.2 ± 19.5/40 hpf), although this was not statistically significant (p = 0.077). Ki67 staining in the angiotensin II group (161.8 ± 14.9) was similar to control. Representative images of cellular staining at day 54 are shown at 20x magnification in Figure 3.

Figure 3.

Figure 3.

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Ki67 in prostate tumors. Treatment with losartan for 29 days resulted in a reduction of Ki67 at 54 days (A) when compared to controls (B). n = 9/group; *p = 0.039 vs control (ttest). 20× magnification.

AT1R and AT2R gene expression analysis

Losartan treated tumors had numerically higher AT1R and AT2R expression. However, 29 days post-randomization, none of these differences were significant (all p>0.1). Administration of angiotensin II resulted in significantly greater expression of both AT1R and AT2R (5.6 ± 1.5 and 21.9 ± 12.5 fold expression, respectively) when compared to controls, with p = 0.043 and 0.038, respectively.

By day 54, there were no numerical differences in AT1R or AT2R expression between any groups. Gene expression results are illustrated in Figure 4.

Figure 4.

Figure 4.

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Angiotensin receptor gene expression in prostate tumors. Treatment with either losartan or angiotensin II induced expression of AT1R (A) and AT2R (B), with expression of both being highest in the angiotensin II group (n = 4/group; p = 0.043 and 0.038, AT1R and AT2R, respectively vs controls (ttest)). AT1R and AT2R levels were similar across groups after treatment was discontinued (n = 5/group). Ang II = angiotensin II.

DISCUSSION

The RAS may play an important role in cancer progression and metastasis (2–5, 10,16,17, review in 24) via its receptors, AT1R and AT2R. Some studies indicate that angiotensin II, working via AT1R, increases cancer cell proliferation and spread (9–11). Other studies indicate that, via AT2R, angiotensin II impedes cancer progression (10,16). Based on these studies, we hypothesized that manipulation of the RAS may alter PC progression. To test this, we performed a pilot study of mice treated with losartan (an AT1R inhibitor commonly used for hypertension), angiotensin II, or untreated control in a PC xenograft model. We found that losartan resulted in significantly smaller tumors and reduced, though not significant, Ki67 expression versus control. Although the angiotensin II treated mice had smaller tumors than control, this difference did not reach significance. From these data, we conclude that AT1R blockade, perhaps in combination with AT2R stimulation may have a novel role in PC management. Further preclinical validation, using larger sample sizes, is necessary.

Our findings support the role of AT1R in PC biology, in that the potent AT1R inhibitor, losartan, significantly slowed tumor growth. We do not disregard the observational studies that suggest ARB usage may increase the risk of new cancer diagnosis (12–14), but no causality can be determined because these studies are subject to bias by indication (i.e. the people who took ARBs were fundamentally different in that they needed an ARB, and it is this difference—not the ARB itself—that translated into higher cancer risk). Moreover, another study found no link between ARBs and cancer (15). It is difficult to draw firm conclusions from these observational studies, but when viewed collectively, the preclinical and epidemiological studies, along with findings from our study, suggest that AT1R may be an important therapeutic target in PC.

As described in literature, AT2R has the opposite effect to AT1R on the circulatory system—vascular smooth muscle and blood pressure (25–27). These opposing effects appear to carry into tumor biology. Specifically, AT1R signaling seems to promote cell proliferation and tumor growth, while AT2R signaling inhibits cell proliferation and tumor growth (9,11). Indeed, multiple studies have shown a decrease in AT2R expression in higher grade adenocarcinomas (2,10,17,18, 28–30). Consistent with these findings, we observed that angiotensin II, which stimulates both AT1R and AT2R, has no significant effect on tumor growth. The observation that stimulation of both AT1R and AT2R has no effect on tumor growth is very important, because we found that blockade of AT1R inhibited tumor growth. Thus, one might predict that angiotensin II, working via AT1R, would promote tumor growth. However, this did not occur. We posit that the tumor growth-enhancing effects of AT1R activation were counterbalanced by the growth-inhibiting effects of activated AT2R. If true, our data lend further support to the idea that AT2R has anti-tumor properties and can negate the pro-tumor effects of AT1R.

Interestingly, angiotensin II resulted in significant up-regulation of both AT1R and AT2R with greater than 20 fold expression of AT2R, an effect that was lost once treatment stopped. In the face of both AT1R and AT2R stimulation, the 20-fold increase in AT2R had no net impact on tumor growth, but it is intriguing to think that angiotensin II could be used in combination with an ARB to up-regulate the anti-tumor AT2R while blocking AT1R. These therapeutic manipulations, though feasible in animal models, would be of concern in human trials, where the use of angiotensin II could induce hypertension. A preferable approach would be an AT2R agonist combined with an AT1R antagonist (e.g. losartan). It is unknown whether an AT2R agonist alone would result in comparable up-regulation of AT2R as seen with angiotensin II. Larger preclinical studies are needed to test these interactions.

Like all studies, ours was not devoid of limitations. Our group sizes were small. This was purposeful and done to provide proof-of-principle data that altering the RAS could influence PC growth. Another limitation is that we performed these animal studies using human prostate cancer cell lines that were not endogenous to the test species. The relevance of this limitation to human prostate cancer treatment in humans is unclear and requires further study. Our results, showing losartan inhibition of tumor growth, provide data to support larger more definitive studies. We also hypothesized here that AT1R inhibition with AT2R stimulation may be the best therapeutic approach, but this arm was not included in the current study. It will be a future study focus. The surgically implanted osmotic minipumps, through which angiotensin II was administered, were potential confounders in the comparison of angiotensin II mice to control mice, which had no implanted pumps. However, as losartan was given in the drinking water, there was a direct, unconfounded comparison between the losartan group and controls. Another limitation is that altering the RAS could affect blood pressure, but we did not measure blood pressure in our pilot study. Irrespective of whether there were blood pressure changes in our mouse model, the implications of any such change in regard to humans is unclear at best. Finally, our model was not a hypertensive model. The clinical question, however, is whether losartan—or any alteration of the RAS—can affect PC growth in men who do not otherwise have indication for anti-hypertensives. We specifically chose a non-hypertensive model. It is unknown whether a hypertensive model would produce similar results.

CONCLUSION

Our pilot data support the hypothesis that selective blockade of AT1R is effective in reducing prostate tumor growth. Alternatively, angiotensin II, which activates and induces AT1R and AT2R, had little effect on tumor growth. These data led us to speculate that stimulation of the anti-tumor AT2R may counterbalance the pro-growth simulation of AT1R. However, as we did not conduct AT2R blockade studies as part of this pilot, no firm conclusions can be drawn. Further studies are needed. Based upon our pilot data, we hypothesize that selectively activating and inducing AT2R plus blocking AT1R may slow PC growth. Our findings support the idea that the RAS is a complex, but targetable pathway in PC. Future larger studies are needed to confirm these results and explore this complexity by testing the inhibition of AT1R coupled with simultaneous AT2R activation as a possible treatment strategy.

ACKNOWLEDGEMENTS

Research was funded by the Durham VA Medical Center, with additional support from the Duke O’Brien Center for Kidney Research (1P30 DK096493-01), NIH (1K24CA160653 and 3R01-CA125618-08S1), Duke University School of Medicine, and The Stewart Rahr–Prostate Cancer Foundation.

Abbreviations:

PC

Prostate cancer

RAS

Renin-Angiotensin System

AT1R

Type 1 Angiotensin Receptor

AT2R

Type 2 Angiotensin Receptor

Footnotes

DISCLOSURES AND CONFLICTS OF INTEREST

Animal studies were conducted at the Durham VA Medical Center (Durham, NC); other studies were conducted at the Division of Nephrology, Department of Medicine, Duke O’Brien Center for Kidney Research, and Division of Urology, Department of Surgery, Duke University Medical Center (Durham, NC). Statistics were provided by the Department of Biostatistics and Bioinformatics, Duke University School of Medicine.

All animal procedures were IACUC approved and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (ILAR, 2011).

None of the authors has financial conflicts of interest regarding this research, nor are any of the authors affiliated or involved, directly or indirectly, with any entity having a financial interest in this research.

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