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. 2015 May 27;29(9):3726–3736. doi: 10.1096/fj.14-267799

Inhibition of hyperglycemia-induced angiogenesis and breast cancer tumor growth by systemic injection of microRNA-467 antagonist

Irene Krukovets 1, Matthew Legerski 1, Pavel Sul 1, Olga Stenina-Adognravi 1,1
PMCID: PMC4550367  PMID: 26018675

Abstract

Abnormal angiogenesis in multiple tissues is a key characteristic of the vascular complications of diabetes. However, angiogenesis may be increased in one tissue but decreased in another in the same patient at the same time point in the disease. The mechanisms of aberrant angiogenesis in diabetes are not understood. There are no selective therapeutic approaches to target increased neovascularization without affecting physiologic angiogenesis and angiogenesis in ischemic tissues. We recently reported a novel miRNA-dependent pathway that up-regulates angiogenesis in response to hyperglycemia in a cell- and tissue-specific manner. The goal of the work described herein was to test whether systemic administration of an antagonist of miR-467 would prevent hyperglycemia-induced local angiogenesis in a tissue-specific manner. We examined the effect of the antagonist on hyperglycemia-induced tumor growth and angiogenesis and on skin wound healing in mouse models of diabetes. Our data demonstrated that the systemic injection of the antagonist prevented hyperglycemia-induced angiogenesis and growth of mouse and human breast cancer tumors, where the miR-467 pathway was active in hyperglycemia. In tissues where the miR-467–dependent mechanism was not activated by hyperglycemia, there was no effect of the antagonist: the systemic injection did not affect skin wound healing or the growth of prostate tumors. The data show that systemic administration of the miR-467 antagonist could be a breakthrough approach in the treatment and prevention of diabetes-associated breast cancer in a tissue-specific manner without affecting physiologic angiogenesis and angiogenesis in ischemic tissues.—Krukovets, I., Legerski, M., Sul, P., Stenina-Adognravi, O. Inhibition of hyperglycemia-induced angiogenesis and breast cancer tumor growth by systemic injection of microRNA-467 antagonist.

Keywords: extracellular matrix, thrombospondin, vascular complications, diabetes, neovascularization

According to Centers for Disease Control (CDC, Atlanta, GA, USA) reports, vascular complications are the main cause of mortality and hospitalization of patients with both types of diabetes (http://www.cdc.gov/diabetes/). Vascular complications of diabetes are characterized by abnormal angiogenesis, but changes in angiogenesis are tissue- and organ-specific and may result in uncontrolled neovascularization in one organ but deficient angiogenesis in another organ or tissue (1, 2). In addition to classic vascular complications, another disease that is critically dependent on angiogenesis—cancer—is also associated with diabetes in a tissue-specific manner. Breast, liver, and pancreatic cancers are positively associated with diabetes, whereas prostate cancer demonstrates a negative association, as has been documented in multiple clinical studies. The mechanisms accounting for the tissue-specific aberrant angiogenesis in diabetes are not understood; however, hyperglycemia has been identified as an independent cause of vascular complications.

In our previous publications, we have described a novel mechanism of tissue-specific regulation of angiogenesis in response to hyperglycemia (4, 5). We reported that high glucose in cell culture and hyperglycemia in vivo stimulate the increased production of miR-467 in a cell-type– and tissue-specific manner. In vivo, miR-467 is up-regulated in tissues that are associated with increased angiogenesis in patients with diabetes. The effect of miR-467 is mediated by thrombospondin (TSP)-1: miR-467 binds to the 3′-UTR of TSP-1 mRNA and suppresses TSP-1 mRNA translation and the production of TSP-1. TSP-1 is a potent endogenous inhibitor of angiogenesis, and a decrease in its levels causes an increase in angiogenesis in response to up-regulated miR-467 levels. We have described the molecular features of this novel miRNA-dependent mechanism (5, 6). The goal of the study described herein was to find out whether the novel miR-467 mechanism of regulation of angiogenesis is operative in vivo and whether hyperglycemia-induced angiogenesis would be prevented in a tissue-specific manner by systemic administration of an miR-467 antagonist. In this work, we used mouse models of tumor angiogenesis and skin wound healing to demonstrate that systemically administered miR-467 antagonist decreases hyperglycemia-induced angiogenesis without negative effects on angiogenesis in tissues that are not affected by hyperglycemia-induced excessive neovascularization. The overall goal of the study was to explore an attractive novel therapeutic target that can be easily and safely modulated to prevent neovascularization in diabetes and the vascular complications caused by excessive angiogenesis.

MATERIALS AND METHODS

Animals

All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee and in agreement with the National Institutes of Health (NIH, Bethesda, MD, USA) Guide for the Care and Use of Laboratory Animals. Leprdb/db (animal model of type 2, non–insulin-dependent, diabetes), Leprdb/+, BALBc, nude, and wild-type C57BL/6 were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Induction of hyperglycemia

In male BALBc and nude C57Bl6 mice, hyperglycemia was induced by streptozotocin (STZ) injections according to the Jackson Laboratory protocol and as described in our publication (5). Citric buffer was injected to control normoglycemic mice. One week after the first STZ injection, blood glucose was measured with an Alphatrak glucometer and blood glucose strips for mice and rats from Abbott (Abbott Park, IL, USA), and mice with levels ≥250 mg/dl were included in the experiments. STZ-induced hyperglycemia is an animal model of type 1, insulin-dependent diabetes.

Cancer cell lines

Cells of the mouse EMT6 and Ac711 lines and of the human MDA-MB-231 line were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The mouse MMTV-Wnt-1 cell line was a kind gift of Dr. Stephen Hursting (Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, TX, USA) (6). EMT6, Ac711, and MDA-MB-231 cells were cultured according to ATCC directions. MMTV-Wnt-1 cells were propagated in vivo in wild-type C57Bl6 mice (Jackson Laboratory) and were harvested (7) and frozen in aliquots at −80°C.

Injection of cancer cells and harvest of tumors

Cancer cells were injected into the mammary fat pad of 12-wk-old Leprdb/db mice or 9-wk-old STZ-treated mice (immediately after hyperglycemia ≥250 mg/dl was documented, 1 wk after the beginning of STZ injections) as described in our prior report (5). On the day of an experiment, cultured cells were harvested or frozen cells were thawed, viable cells were counted, and cancer cells were injected into the mammary fat pad: EMT6, 1.5 × 106 cells in 100 μl PBS; Ac711, 1.5 × 106 cells in 100 μl PBS; MDA-MB-231, 8 × 106 cells in 100 μl Matrigel; and MMTV-Wnt-1, 1 × 106 cells in 100 μl PBS. Tumors were harvested when the largest tumors (in hyperglycemic mice) reached the maximum allowed size (1.7 mm3), as follows: EMT6 on d 11, Ac711 on d 18, MDA-MB-231 on d 11, and MMTV-Wnt-1 on d 19. Murine prostate carcinoma RM1 cells (1.5 × 106) were injected into 9-wk-old wild-type C57Bl/6 mice, and the tumors were harvested on d 9 when the largest tumors reached the maximum size allowed under NIH animal use guidelines.

Mice were euthanized by CO2 inhalation, followed by cervical dislocation. Tumors were excised, photographed, weighed, and frozen in optimal cutting temperature compound (Tissue-Tek; Sakura Finetek, Inc., Torrance, CA, USA) in liquid nitrogen and stored at −80°C.

Injections of miR-467 antagonist

The locked nucleic acid (LNA)–modified miR-467 antagonist 5′-TacaTGcaGGcacTTa and the control oligonucleotide 5′-TTTaGaccgaGcgTGt (LNA-modified bases are shown in capital letters) were synthesized by Exiqon (Woburn, MA, USA). The control oligonucleotide does not have any predicted targets and has been used before in our work (6).

The oligonucleotides were reconstituted in PBS, divided into aliquots, and stored at −20°C. They were injected at 2.5 mg/kg body weight i.p. on d 3 after cancer cell injection; on d 8; and, if tumors were grown for more than 12 d, once a week thereafter (Ac711 and MMTV-Wnt-1). In the wound-healing experiments, oligonucleotides were injected 1 d before wounding and then on d 3 and 8.

Stimulation of miR-467 production by high-glucose Ac711 cells

To cultured MDA-MB-231, Ac711, human neonatal normal epithelial keratinocytes (HNEKs; Lonza, Walkersville, MD, USA), and human foreskin fibroblasts (HFFs; ATCC, Manassas, VA, USA), 5 mM d- or l-glucose was added after the cells were preincubated overnight in low-glucose DMEM (Cellgro; Corning Life Sciences, Manassas, VA, USA). The cells were washed and harvested by scraping, and RNA was immediately isolated with TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). RNA (1 mg) was used to detect miR-467 by quantitative (q)RT-PCR as described earlier in (6). 5s RNA was used for normalization.

Immunohistochemistry

Sections (10 μm) were processed (5) and stained with antibodies against cluster of differentiation (CD)31 (BD Pharmingen, San Jose, CA, USA), laminin (Abcam, Cambridge, MA, USA), and α-actin (Abcam). Images of stained sections were acquired with a DMR 4000B upright microscope (Leica, Heidelberg, Germany) fitted with a single-slide x-, y-, and z-motorized stage, a ×20 (dry) objective, an FITC fluorescence filter cube, and a Retiga 2000R charge-coupled device (CCD) digital camera (QImaging, Burnaby, BC, Canada). For quantitative analysis, images were processed with Image-Pro Plus 6.1 (Media Cybernetics, Silver Spring, MD, USA). The total area of the section, percentage of stained area, and intensity of staining of each cross-section were exported to Excel (Microsoft, Redmond, WA, USA). Levels of angiogenesis markers are presented in the figures as the average percentage of stained area of section × tumor mass, to reflect the total tumor angiogenic response. Differences in percentage of stained area per section between antagonist-treated and control tumors were statistically significant without normalization to tumor mass in most cases, with the exception of laminin-1 values in Ac711 and EMT6 cells in Leprdb/db mice and α-actin values in EMT6 tumors in BALBc mice, which became significant only after normalization to tumor mass. Intensity of staining was used to ensure that similar average staining intensity was selected to quantify the angiogenesis markers in evaluating the area of vessel coverage.

TSP-1 levels are expressed in the figures as the average percentage of stained area of the cross-section × average intensity of staining, because TSP-1 is a secreted protein, and its levels are reflected in both the area of staining and the intensity of staining (both of which change in response to hyperglycemia).

Glucose and insulin tolerance tests

After 12 h of fasting, 2 g/kg glucose or 50 μg/kg insulin (both from Sigma-Aldrich, St. Louis, MO, USA) was injected intraperitoneally. Blood glucose was measured in blood samples from tail vein before glucose or insulin injection and then 15, 30, 60, 90, and 120 min after injection with an Alphatrak glucometer and blood glucose strips for mice and rats (Abbott).

Skin wound-healing model

Leprdb/db mice were injected with miR-467 antagonist 1 d before wounding, to assess the effect of pre-existing blood levels of antagonist on wound healing, as it would be relevant in a potential clinical situation. Single excisional wounds of 0.5 cm diameter were made on the back of the mouse and traced, photographed, and measured on the day of surgery and on d 1, 3, 7, 10, 11, 13, and 14 thereafter. The intraperitoneal injections of antagonist and control oligonucleotide were given 1 d before the surgery and again on d 3 and 7, to match the injection schedule of the experiments in the cancer models described above. The wounds completely healed by d 13. The area of each wound was measured with Photoshop 8 (Adobe, San Jose, CA, USA), excised, and frozen at the end of the experiment. Sections of the skin were stained for the angiogenesis markers CD31, α-actin, laminin-1, and TSP-1, as described above.

Statistical analysis

All results are expressed as means ± sem. Before an analysis, the Shapiro-Wilk normality test was used on each data set, on the original scale or after transformation, and parametric methods were used when the assumption of normality was met, whereas nonparametric methods were applied if the assumption was violated. Two- or 1-way ANOVA was used for data with 2 factors or 1, respectively, and Student’s t test was used to compare 2 group means. The nonparametric counterparts were the Friedman rank sum test with stratifications for 2-way ANOVA, the Kruskal-Wallis test for 1-way ANOVA, and the Wilcoxon rank-sum test for the t test. For subgroup multiple comparisons, Tukey’s test was applied. For correlation analysis, Spearman’s rank correlation test was used for its relaxed assumptions of normality and linearity. The results were considered statistically significant at α = 0.05. All data analyses were performed with the statistical software package R (R Development Core Team,http://www.R-project.org).

RESULTS

Inhibition of EMT6 tumor growth and vascular remodeling in Leprdb/db mice by systemic injection of miR-467 antagonist

We have reported that the growth of breast tumors in EMT6 cell-induced cancer is dramatically accelerated in male Leprdb/db mice (5). Although breast cancer affects both males and females, it is more frequent in females. In our experiments, we used both sexes. We confirmed that EMT6 tumor growth in the female Leprdb/db mice (C57Bl/6 background) is dramatically affected by hyperglycemia to the same extent as in male mice (Fig. 1A and Supplemental Fig. 1A, left).

Figure 1.

Figure 1.

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Effect of an miR-467 antagonist on mouse EMT6 and Ac711 cell-induced breast tumor growth in Leprdb/db mice. A) Effect of hyperglycemia on EMT6 tumor growth. Cancer cells (1.5 × 106) in 100 μl PBS were injected into the mammary fat pad of female (C57BL/6 background, hyperglycemic) and heterozygous control (normoglycemic) mice. Tumors were harvested on d 11. The results are presented as the mean tumor mass ± sem. #P < 0.05 vs. normoglycemic mice (n = 10 in each group). B) Effect of miR-467 antagonist injection on EMT6 tumor growth. Cancer cells were injected into hyperglycemic male mice and normoglycemic controls, as described in (A). Mice received antagonist or control oligonucleotide injected intraperitoneally on d 3 and 7. Tumors were harvested on d 11. The results are presented as mean tumor mass ± sem. #P < 0.05 vs. normoglycemic group; *P < 0.05 vs. control oligonucleotide-injected group (n = 10, each group). C) Effect of antagonist injection on levels of the angiogenesis markers CD31, laminin-1, and α-actin in the tumors. Proteins were detected by immunohistochemical staining and quantified as the mean stained area (%) × tumor mass ± sem. *P < 0.05 vs. control- oligonucleotide-injected mice (n = 10, each group). D) Effect of the antagonist on Ac711 cell tumor growth. Cancer cells were injected into hyper- and normoglycemic mice as described in (A). On d 3 after injection, mice received an injection (2.5 mg/kg i.p.) of antagonist or control oligonucleotide, followed by additional injections on d 7 and 12. Tumors were harvested on d 19. The results are presented as the mean tumor mass ± sem. #P < 0.05 vs. normoglycemic mice; *P < 0.05 vs. control oligonucleotide-injected mice (n = 5, each group). Most tumors were not detectable in the control group. E) Effect of antagonist injection on levels of angiogenesis markers CD31, laminin-1, and α-actin in the tumors. The proteins were detected and quantified as described in (C). *P < 0.05 vs. the control oligonucleotide-injected group (n = 5, each group). F) Increased levels of miR-467 in cultured Ac711 cancer cells in response to high glucose. Cells were incubated in low-glucose medium 24 h before stimulation. d- or l-Glucose (25 mM) was added to the medium for 48 h, the cells were harvested, RNA was isolated, and miR-467 levels were quantified by real-time qRT-PCR (RQ). Data are expressed as the mean level in high (25 mM) glucose samples/mean level in control (5 mM) glucose samples ± sem. *P < 0.05 vs. the low-glucose control (n = 3).

To observe the effect of the systemic administration of the miR-467 antagonist, we injected 1.5 × 106 cancer cells in 100 μl PBS into the mammary fat pad of 12-wk-old female Leprdb/db and heterozygous control normoglycemic mice 3 d before the injection of the antagonist. On d 3, when the growth was detectable by palpation, we injected antagonist or control oligonucleotide (2.5 mg/kg i.p.), followed by another injection on d 7. Tumors were harvested on d 11 when the largest ones reached ∼1.5 cm3. The antagonist had a dramatic effect on tumor growth: the average weight of tumors decreased by 3-fold compared with the average weight in mice injected with the control oligonucleotide (Fig. 1B and Supplemental Fig. 1A, right).

To evaluate the effect of the antagonist on vascular remodeling in tumors, we detected and quantified CD31, a marker of endothelial (ECs); laminin-1, a basement membrane marker that serves as an indicator of a formed vessel; and α-actin, a marker of smooth muscle cells and pericytes (Fig. 1C and Supplemental Fig. 2). Taken together, these 3 markers characterize the angiogenesis process, migration of ECs into the tissue, and vessel formation and maturation.

The proteins were detected with specific antibodies in frozen tissue sections of EMT6 tumors grown in hyperglycemic Leprdb/db mice that received either the control oligonucleotide or the miR-467 inhibitor, and the images were quantified by a blinded investigator, as described in Materials and Methods. The amounts of all 3 markers in the tumor sections were decreased in hyperglycemic animals that received the antagonist, when compared with animals that received the control oligonucleotide (Fig.1C), consistent with the growth of the tumors in these 2 conditions.

Inhibition of Ac711 tumor growth in Leprdb/db mice by systemic injection of miR-467 antagonist

To confirm that the effect of miR-467 is not limited to a single cancer cell line, we used 2 other mouse breast cancer lines—Ac711 (8–11) and MMTV-Wnt-1 (12)—to induce tumors in Leprdb/db mice. Ac711 cells (1.5 × 106) were injected into the mammary fat pad, and the first injection of miR-467 was given on d 3 after the cancer cell injection. The second injection was given on d 7, and the third one on d 12 (the tumors grew slower than the EMT6 tumors). The tumors were harvested on d 18. The Ac711 tumors were 30 times larger in Leprdb/db mice (0.98 ± 0.13 g) than those in Leprdb/+ mice (0.03 ± 0.02 g) (Fig. 1D and Supplemental Fig. 1B). Antagonist injections significantly reduced tumor growth in Leprdb/db mice (to 0.48 ± 0.19; P = 0.03).

The levels of the angiogenesis markers CD31, laminin-1, and α-actin were quantified in hyperglycemic mice that received the antagonist or the control oligonucleotide. The amounts of all 3 markers in the tumor sections were decreased in hyperglycemic animals that received the antagonist, as compared with animals that received the control oligonucleotide (Fig. 1E), consistent with the growth of the Ac711 tumors in these 2 conditions. Similar to EMT-6 cancer cells, an increased level of miR-467 was detected in cultured Ac711 cells in response to 25 mM glucose (Fig. 1F).

Inhibition of MMTV-Wnt-1 tumor growth in Leprdb/db mice by systemic injection of miR-467 antagonist

Increased growth of MMTV-Wnt-1 cell-induced tumors in Leprdb/db mice has been reported (5, 7). We compared the growth of MMTV-Wnt-1 tumors in Leprdb/db mice treated with the miR-467 antagonist and Leprdb/db mice treated with the control oligonucleotide (Fig. 2A and Supplemental Fig. 1C). As in the experiments described above, the first injection was given on d 3 after the cancer cell injection, followed by a second injection on d 7, and a third one on d 12. Tumors were harvested on d 19. The antagonist significantly inhibited MMTV-Wnt-1 tumor growth in Leprdb/db mice: 0.24 ± 0.03 g in mice injected with the antagonist vs. 0.69 ± 0.09 g in mice injected with the control oligonucleotide (P = 0.003).

Figure 2.

Figure 2.

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Effect of miR-467 antagonist on mouse MMTV-Wnt-1 and human MDA-MB-231 breast cancer tumor growth in Leprdb/db and NU/J mice, respectively. A) Effect of antagonist on MMTV-Wnt-1 tumors. Cancer cells (1 × 106) in 100 μl PBS were injected into the mammary fat pad of the mice. On d 3 after the injection, a 2.5 mg/kg i.p. injection of the antagonist or control oligonucleotide was given, followed by additional injections on d 7 and 12. Tumors were harvested on d 19 (n = 8, control group; n = 10, antagonist group). The results are presented as mean tumor mass ± sem. #P < 0.05 vs. normoglycemic; *P < 0.05 vs. control oligonucleotide-injected group (n ≥ 8, each group). B) Effect of antagonist injection on levels of the angiogenesis markers CD31, laminin-1, and α-actin in the tumors. Proteins were detected by immunohistochemical staining, and the protein levels were quantified as mean stained area (%) × tumor mass ± sem. *P < 0.05 vs. control oligonucleotide-injected group (n = 8, control group; n = 10, antagonist group. C) Effect of the antagonist on human MDA-MB cancer tumor growth in immunodeficient NU/J mice. Cancer cells (8 × 106) in 100 μl PBS were injected into the mammary fat pad of STZ-treated or untreated NU/J mice. On d 3 after the cancer cell injection, a 2.5 mg/kg i.p. injection of antagonist or control oligonucleotide was given, followed by a second injection on d 7. Tumors were harvested on d 11. The results are presented as mean tumor mass ± sem. #P < 0.05 vs. normoglycemic mice; *P < 0.05 vs. control oligonucleotide-injected group (n = 4–10 per group; Supplemental Fig. 1). The difference between the normoglycemic and hyperglycemic antagonist groups is also statistically significant (P = 0.026). D) Effect of antagonist injections on levels of the angiogenesis markers CD31, laminin-1, and α-actin in the breast tumors in immunodeficient NU/J mice. Proteins were detected and quantified as described in (B). *P < 0.05 vs. control oligonucleotide-injected group (n = 4–10 per group; Supplemental Fig. 1). E) Increased levels of miR-467 in cultured MDA-MB-231 cancer cells in response to high glucose. Cells preincubated in low-glucose medium (5 mM d-glucose) were treated for 48 h with 5 mM d-glucose (control) or 25 mM d- or l-glucose. RNA was extracted from the cells, and miR-467 was quantified by real-time qRT-PCR (RQ). Data are expressed as the mean level in high-glucose (25 mM) samples/mean level in control 5 mM glucose samples ± sem. *P < 0.05 vs. the control (n = 3).

The levels of the angiogenesis markers CD31, laminin-1, and α-actin were quantified in hyperglycemic mice that received the antagonist or the control oligonucleotide. The amounts of all 3 markers in the tumor sections were decreased in hyperglycemic animals that received the antagonist, when compared with the levels in animals that received the control oligonucleotide (Fig. 2B), consistent with the growth of the MMTV-Wnt-1 tumors in these conditions.

Inhibition of MDA-MB-231 tumor growth in STZ-treated hyperglycemic mice by systemic injection of miR-467 antagonist

We tested the effect of systemic injection of miR-467 on in vivo growth of human MDA-MB-231 cell-induced tumors in immunodeficient nude NU/J mice (Fig. 2C and Supplemental Fig. 1D). Cells (8 × 106) were injected with 100 μl Matrigel (Corning Life Sciences), followed by injections of the antagonist on d 3 and 7. Tumors were harvested on d 11. Similar to the mouse cancer cell lines, human cancer tumor growth in vivo was accelerated by hyperglycemia and inhibited by the antagonist injection. The average tumor weight in hyperglycemic mice that received the antagonist was 0.053 ± 0.003 vs. 0.13 ± 0.017 in mice that received the control oligonucleotide (P = 0.003).

The levels of the angiogenesis markers CD31, laminin-1, and α-actin were quantified in hyperglycemic mice that received the antagonist or the control oligonucleotide. The amounts of all 3 markers in the tumor sections were decreased in hyperglycemic animals that received the antagonist, as compared with animals that received the control oligonucleotide (Fig. 2D), consistent with the growth of the MDA-MB-231 tumors in these 2 conditions. Cultured human MDA-MB-231 breast cancer cells expressed increased amounts of miR-467 in response to high glucose (Fig. 2E), similar to the mouse breast cancer cell lines EMT6 (5) and Ac711.

Inhibition of EMT6 tumor growth in STZ-treated hyperglycemic BALBc mice by systemic injection of miR-467 antagonist

We have reported that tumor growth and angiogenesis increase in STZ-treated hyperglycemic mice (5). The EMT6 cell line originated in BALBc mice (13). To ensure that the effect of the antagonist on the immune system was not the reason for the decreased tumor growth in C57Bl/6 mice, we examined the effect of the miR-467 antagonist in BALBc mice.

As shown in Fig. 3A and Supplemental Fig. 1E, tumor growth was accelerated by hyperglycemia (0.26 ± 0.05 g in STZ-treated hyperglycemic mice vs. 0.14 ± 0.02 g in normoglycemic mice; P = 0.013) and was efficiently prevented by the antagonist (0.07 ± 0.006 g; P = 0.0002).

Figure 3.

Figure 3.

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Effect of miR-467 antagonist on mouse EMT6 breast cancer tumor growth and angiogenesis in STZ-treated BALBc mice. A) Cancer cells (1.5 × 106) were injected in 100 μl PBS into the mammary fat pad of STZ-treated (hyperglycemic) and control (normoglycemic) mice. On d 3 after the cancer cell injection, an injection (2.5 mg/kg i.p.) of antagonist or control oligonucleotide was given, followed by a second injection of on d 7. Tumors were harvested on d 11. The results are presented as mean tumor mass ± sem. #P < 0.05 vs. control (normoglycemic) mice; *P < 0.05 vs. control oligonucleotide-injected group; (n = 8–10 per group; Supplemental Fig. 1). B) Effect of antagonist injection on levels of the angiogenesis markers CD31, laminin-1, and α-actin in 10 μm sections of tumors. The markers were detected by immunohistochemical staining, and expression was quantified as mean stained area (%) × tumor mass ± sem. *P < 0.05 vs. control oligonucleotide-injected group (n = 8–10 per group; Supplemental Fig. 1). C) Effect of antagonist on TSP-1 protein levels and tumor mass. Sections (10 μm) were stained with anti-TSP-1 antibody and quantified as the mean stained area (%) × mean intensity of staining ± sem. *P < 0.05 vs. control oligonucleotide-injected group. Levels of TSP-1 were quantified as mean stained area (%) × tumor mass ± sem. *P < 0.05 vs. control-injected group (N as described above in figure legends for experiments with corresponding cancer cell lines).

The levels of the angiogenesis markers CD31, laminin-1, and α-actin were quantified in hyperglycemic STZ-treated BALBc mice that received the antagonist or the control oligonucleotide. The amounts of all 3 markers in the tumor sections were decreased in hyperglycemic animals that received the antagonist as compared with animals that received the control oligonucleotide (Fig. 3B), consistent with the growth of the EMT6 tumors in BALBc mice in these 2 conditions.

The injections of the antagonist also decreased the average size of tumors in normoglycemic mice (0.13 ± 0.07 g in normoglycemic mice injected with control oligonucleotide vs. 0.057 ± 0.023 in normoglycemic mice injected with miR-467 antagonist; P = 0.0018), but the levels of the angiogenesis markers were not changed by the antagonist in normoglycemic mice (not shown).

The effect of miR-467 antagonist on TSP-1 level in tumors

We have reported that miR-467 downregulates TSP-1 mRNA translations in response to high glucose, and that the miR-467 antagonist restores the production of TSP-1 (5).

We examined the levels of TSP-1 in tumors from hyperglycemic mice that received the control oligonucleotide and miR-467 antagonist (Fig. 3C). The area of staining in the sections of all tumors described above and the intensity of staining were quantified, and the results are presented as stained area × intensity of staining. In all breast tumors, the level of TSP-1 was up-regulated by the antagonist injection, and in MMTV-Wnt-1 tumors grown in Leprdb/db mice, in MDA-MB-231 grown in NU/J mice, and in EMT6 tumors grown in BALBc mice, the difference was statistically significant (P < 0.05).

Systemic injection of miR-467 antagonist does not affect glucose and insulin tolerance

Fasting glucose levels in 4 mouse strains used in this study (Leprdb/db, NU/J, BALBc, and C57BL/6) measured before the injection of cancer cells are shown in Fig. 4. In all strains, with the exception of Leprdb/db, fasting glucose levels at the start of the experiment were ≥250 mg/dl but <300 mg/dl.

Figure 4.

Figure 4.

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Systemic injection of miR-467 antagonist does not affect the metabolism of glucose. A) Fasting blood glucose levels at the beginning of the experiments were measured in mice after overnight fasting (14 h) and are expressed as mean blood glucose levels ± sem. *P < 0.05 vs. control oligonucleotide-injected group (N as described above in figure legends for experiments with corresponding cancer cell lines). B, C) Lack of effect of the antagonist on glucose level and metabolism. Glucose (B) and insulin (C) tolerance tests were administered to 14-wk-old Leprdb/db mice after 2 mo of weekly injections of the antagonist. The results are presented as mean blood glucose levels ± sem (n = 5).

To understand whether the metabolic changes caused the decrease in breast cancer tumor growth in mice injected with the antagonist, we administered glucose and insulin tolerance tests to 14-wk-old Leprdb/db mice after 8 wk of weekly injections. In mice that received injections of the antagonist, there was no change in fasting blood glucose levels or 15, 30, 60, 90, and 120 min after the intraperitoneal injection of glucose (Fig. 4B) or insulin (Fig. 4C).

Systemic injection of miR-467 antagonist does not affect prostate tumor growth

We reported that mouse prostate cancer cells do not respond to high glucose by increasing miR-467 in culture and that hyperglycemia does not accelerate prostate tumor growth in a mouse model (5). These observations are consistent with the decreased incidence of prostate cancer in patients with diabetes reported in multiple clinical studies. To confirm that the effect of the miR-467 antagonist on angiogenesis is tissue specific, we examined mouse RM1 prostate cancer tumor growth and wound healing in hyperglycemic mice. RM1 cells (1.5 × 106) were injected subcutaneously into STZ-treated male C57Bl/6 mice, and the injections of oligonucleotides were given on d 3, followed by a second injection on d 7. Tumors were collected on d 9.

RM1 tumors were smaller in STZ-treated hyperglycemic mice (Fig. 5A and Supplemental Fig. 1F), consistent with the lack of miR-467 up-regulation by glucose in the cells and lack of inhibition of TSP-1 production (5). When the antagonist was administered to hyperglycemic mice injected with RM1 cancer cells in an experiment identical with those described above, there was no effect on tumor weight.

Figure 5.

Figure 5.

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Lack of effect of the miR-467 antagonist on mouse RM1 prostate cancer tumor growth and on wound healing in hyperglycemic STZ-treated C57BL/6 mice. A) Cancer cells (1.5 × 106 ) in 100 μl PBS were injected subcutaneously into male STZ-treated (hyperglycemic) or untreated control (normoglycemic) mice. On d 3 after the cancer cell injection, an injection (2.5 mg/kg i.p.) of antagonist or control oligonucleotide was given, followed by additional injection on d 7. Tumors were harvested on d 9. The results are presented as mean mass ± sem. #P < 0.05 vs. normoglycemic mice (n = 4–5 per group; Supplemental Fig. 1B). Effect of antagonist injection on levels of the angiogenesis markers CD31, laminin-1, and α-actin in the tumors. The proteins were detected by immunohistochemical staining, and the protein levels were quantified as mean stained area (%) × tumor mass ± sem (n = 4–5 per group; Supplemental Fig. 1). C) Effect of miR-467 antagonist injection on excisional skin wound healing in Leprdb/db mice. Antagonist or control oligonucleotide was injected (2.5 mg/kg i.p.) into mice 1 d before a single excisional skin wound was inflicted, followed by additional injections on d 3 and 7. Wounds were measured on the days indicated. The results are presented as mean wound area (pixels) ± sem. (n = 10). D) Effect of antagonist injection on levels of the angiogenesis markers CD31, laminin-1, and α-actin in the excisional skin wound-healing model. The proteins were detected by immunohistochemical staining, and levels were quantified as the mean stained area (%) ± sem (n = 10, in each group). E) Levels of miR-467 in cultured HNEKs and HNEKs in response to high glucose. Cells were incubated in low-glucose medium 24 h before stimulation. d- or l-Glucose (25 mM) was added to the medium for 48 h, the cells were harvested, RNA was isolated, and miR-467 levels were quantified by real-time qRT-PCR (RQ). Results are presented as mean level in high-glucose (25 mM) samples/mean level in control 5 mM glucose samples ± sem. *P < 0.05 vs. control (n = 3).

We examined the angiogenesis markers CD31, laminin-1, and α-actin in RM1 tumors from hyperglycemic mice injected with the control oligonucleotide and miR-467 antagonist (Fig. 5B). We were surprised that all markers tended to decrease. However, the decrease in the levels of either of the 3 markers did not reach statistical significance.

Systemic injection of miR-467 antagonist does not affect wound healing

The effect of the miR-467 antagonist on skin wound healing was assessed in Leprdb/db mice, as described in Materials and Methods. There was no difference in wound size and speed of wound healing between mice injected with the antagonist and mice injected with the control oligonucleotide (Fig. 5C).

CD31 and α-actin levels were quantified in sections of the newly formed skin after the wound healed (Fig. 5D). No difference was detected between the levels of angiogenesis markers in mice that received the antagonist and those that received the control oligonucleotide. The levels of TSP-1 were similar in the groups.

HNEKs and HFFs were cultured according to the providers’ directions and stimulated with 25 mM d- and l-glucose for 48 h, and the level of miR-467 was quantified (Fig. 5E). miR-467 levels were not affected by glucose stimulation, but the keratinocytes responded to both d- and l-glucose by up-regulating the levels of miR-467, similar to the response of microvascular ECs and retinal pigment epithelial cells (5).

DISCUSSION

In this study, hyperglycemia-induced increased angiogenesis was prevented by systemic injection of the antagonist of miR-467 in a tissue-specific manner and without negative effects on angiogenesis-dependent processes in tissues unaffected by high-glucose–induced neovascularization.

Tissue-specific regulation of angiogenesis is a highly desirable, yet not easily achievable, clinical goal. The use of all current antiangiogenesis drugs is limited by their side effects on physiologic angiogenesis that prevent normal remodeling of tissues (e.g., in wound healing and in reproduction). This limitation in the use of currently available antiangiogenesis drugs is even more restrictive in patients with diabetes, because in many of their tissues, angiogenesis is already decreased, and these tissues (e.g., skin and heart) are already affected by ischemia. We have recently described molecular details of a novel tissue-specific pathway of regulation of angiogenesis in hyperglycemia (4, 5). Herein, we describe the activity of this pathway in vivo and its successful and efficient targeting to inhibit hyperglycemia-induced angiogenesis in a tissue-specific manner. The central regulator of this novel pathway is miR-467, which inhibits production of TSP-1, one of the most potent endogenous antiangiogenic agents (4, 5). miR-467 is normally poorly expressed or undetectable, but is highly up-regulated in a cell- and tissue-specific manner in response to high glucose in cultured cells and in vivo in hyperglycemic animals (5). Thus, we hypothesized that targeting miR-467 would not affect the tissues that do not express miR-467 in response to hyperglycemia, but would decrease hyperglycemia-induced angiogenesis in tissues with increased neovascularization and up-regulated miR-467.

Emerging evidence suggests that the incidence of several cancers (e.g., hepatic, pancreatic, colon, endometrial, breast, and bladder cancer) is increased in patients with diabetes and that their prognosis is worse than that of normoglycemic patients (14–18) [reviewed in (19)]. One phenomenon that suggests clues to potential mechanisms by which hyperglycemia regulates the progression of these cancers is the negative correlation of some cancers (e.g., prostate) with diabetes (19–26). Although surprising at first glance, the tissue-specific association of tumor growth with diabetes may result from the well-known tissue-specific regulation of angiogenesis in patients with diabetes. Aberrant angiogenesis of diabetes (increased neovascularization in some tissues, e.g., retina and kidney, and deficient angiogenesis causing ischemia in others, e.g., skin and myocardium) has been recognized for many years and is the cause and hallmark of the vascular complications of diabetes (retinopathy, neuropathy, cardiomyopathy, and nephropathy) (1–3).

In this study, the increased breast tumor growth in hyperglycemic mice was associated with increased angiogenesis: an increase in CD31, a marker of (ECs); in laminin-1, a marker of vascular basement membrane; and in α-actin, a marker of vascular smooth muscle cells and pericytes. Our previous report and the new data from the current study documented dramatically increased angiogenesis and larger breast cancer tumors in hyperglycemic animals in both the mouse model of insulin-dependent type 1 diabetes model (STZ-treated mice) and in the mouse model of non–insulin-dependent type 2 diabetes (Leprdb/db mice).

As a model of a tissue expressing a high level of miR-467 in response to high glucose, we used mouse and human breast cancer tumors grown in hyperglycemic and normoglycemic mice. As a model of tissues that are known to have decreased angiogenesis in diabetes and do not respond to high glucose by increasing miR-467 production, we used prostate tumors (5) and skin, where fibroblasts produce large amounts of TSP-1 in hyperglycemic animals (27).

The antagonist dramatically decreased the growth of several breast cancer cell lines of different origins and with various mechanisms of transformation: mouse cell lines EMT6, Ac711, and MMTV-Wnt-1 and the human cell line MDA-MB-231 (11, 13, 28–32).

In all models, the average weight of tumors was 2 to 3 times lower in hyperglycemic mice treated with the antagonist of miR-467, compared with those in mice treated with the control oligonucleotide that does not have predicted targets in human and mouse genomes. The effect was observed in mouse models of both diabetes types 1 and 2. As is clear from our previous work, high glucose is the stimulus for up-regulation of miR-467 and increased angiogenesis (4, 5).

The precise signaling mechanism of up-regulation of miR-467 is unknown, although we reported that the up-regulation was not mediated by the products of the intracellular glucose metabolism: biologically inactive l-glucose that cannot be transported inside the cells is equally active in inducing miR-467 and in suppressing the production of TSP-1, suggesting increased osmolarity as a stimulus. Hyperosmolarity is a common phenomenon in diabetic animals (33) and humans (34), but its effect on vasculature has not been studied. Hyperosmolar hyperglycemic nonketotic diabetic coma is a frequent complication of diabetes. When the osmolarity of plasma was assessed in groups of younger and older patients with diabetes, the diabetic hyperosmolar state was present in 46% of patients aged <30 yr and in 44% of patients >60 yr of age (34). The damaging effects of this clinically relevant condition have not been well addressed, with a few exceptions [e.g., (35)]. The levels of angiogenesis markers were quantified in all breast cancer tumors and were found to be significantly reduced by injection of an miR-467 antagonist.

The silencing effect of miR-467 on TSP-1 mRNA translation and TSP-1 production, which we described previously (5), suggested that injection of miR-467 antagonist would restore TSP-1 production. Indeed, in all breast tumors grown from different cancer cell lines in mice of different backgrounds, TSP-1 production increased—in most of them, significantly.

Mouse breast cancer cells, several mouse tissues (5), and human breast cancer cells had dramatically increased production of miR-467 in response to high glucose. In contrast to breast cancer cells and to the tissues associated with increased angiogenesis in diabetes, prostate cancer cells and skin fibroblasts do not have increased production of miR-467 in response to high glucose and produce increased amounts of TSP-1 in hyperglycemia because of the increased transcription of TSP-1 gene (5, 27). Thus, the prostate cancer and the wound-healing models were selected to test the tissue specificity of the effects of miR-467 inhibitor. The size of the prostate cancer tumors decreased in hyperglycemic mice, consistent with multiple reports of clinical studies on the decreased incidence of prostate cancer in patients with diabetes. The mechanism for this paradoxical association is unknown. Our results regarding the inactivity of the miR-467 pathway in prostate cancer cells suggest that the decreased incidence of prostate cancer in diabetes is caused by the lack of miR-467 in prostate cancer cells and, as a result, the increased levels of TSP-1 and increased antiangiogenic pressure. There was neither a decrease in tumor size nor an increase in TSP-1 production in response to injection of the antagonist. The levels of angiogenesis markers unexpectedly tended to decrease, although the differences were not statistically significant. The tendency toward decreased TSP-1 levels may be accounted for by activation of the miR-467 pathway in blood vessels of RM1 prostate tumors, but the lack of correlation between the tumor mass and angiogenesis in this model suggests that up-regulation of miR-467 and down-regulation of TSP-1 production have to occur in both the blood vessel wall and the cancer cells, to reduce TSP-1 levels enough to allow sufficient angiogenesis to support tumor growth.

In the wound-healing model, wound size on d 3–5 is dependent on angiogenesis (36–39). In ischemic diabetic tissues (e.g., in skin), decreased angiogenesis is undesirable; the ideal drug for treatment and prevention of vascular complications of diabetes should not decrease angiogenesis in these tissues. As was expected based on our previously published information about the tissue-specific activation of the miR-467 pathway in hyperglycemia, the miR-467 antagonist did not have any effect on healing of excisional skin wounds and did not affect the levels of angiogenesis markers. When cultured skin fibroblasts and epidermal keratinocytes were stimulated with high glucose in culture, they did not respond to glucose stimulation by up-regulation of miR-467, whereas in keratinocytes, miR-467 was up-regulated. This observation suggests that the main source of TSP-1 in skin is fibroblasts, and changes in TSP-1 production in blood vessels or in keratinocytes do not contribute significantly to the total levels of TSP-1 in skin.

Synthetic antagonists of miRNA are stable in vivo and are effective for weeks and even months (2, 40–42). Several miRNA-based prospective therapeutics are currently being examined in preclinical or clinical studies [e.g., Janssen et al. (43), also reviewed in van Rooij et al. (44)]. The data described in this report suggest that the inhibitor of miR-467 has potential as a therapeutic agent with selective effects on diabetic tissues with increased neovascularization. Our results reported previously and the data described herein suggest that the miR-467 antagonist may be a safe inhibitor of hyperglycemia-induced angiogenesis that will not affect physiologic angiogenesis or angiogenesis in ischemic diabetic tissues. Thus, it may represent a new class of selective angiogenesis inhibitors that can be delivered systemically and will exert tissue-specific effects where the control of diabetic neovascularization is desirable.

Supplementary Material

Supplemental Data
supp_29_9_3726__index.html (957B, html)

Acknowledgments

The authors thank Ms. Nadia Hoppe (Department of Slavic Languages and Literatures, University of Illinois at Urbana-Champaign) for technical help with the manuscript preparation and editorial assistance. This work was supported by U.S. National Institutes of Health (NIH) National Heart, Lung, and Blood Institute Grant R01-HL117216, U.S. NIH National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK067532, U.S. NIH National Cancer Institute Grant CA177771, and by funds from the Scott Hamilton Cares Foundation.

Glossary

CD

cluster of differentiation

EC

endothelial cell

HNEK

human neonatal epithelial keratinocyte

HFF

human foreskin fibroblast

LNA

locked nucleic acid

miRNA

microRNA

qRT-PCR

quantitative RT-PCR

STZ

streptozotocin

TSP-1

thrombospondin-1

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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