Suppression of beta 2 adrenergic receptor actions prevent UVB mediated cutaneous squamous cell tumorigenesis through inhibition of VEGF-A induced angiogenesis

Abstract

Although beta 2 adrenergic receptors (β₂ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective β₂ADR antagonists by inhibiting β₂ADR actions in the keratinocytes significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.

1. INTRODUCTION

Among the subtypes of adrenergic receptors (α1, α2, β1, β2 and β3), it is now well established that keratinocytes, the cells that make up the majority of the epidermis, express both α1 and β2 receptors^1–4. Several studies indicate that β2 ADR play essential roles in wound healing and dermatological diseases such as atopic dermatitis, vitiligo and psoriasis^{2, 4}. However, although there are reports demonstrating the effects of carvedilol, a non-specific βADR blocker on skin tumorigenesis, i.e., it acts on all the three β receptors, this drug also has α1 adrenergic activity^5–7. Therefore, it will be interesting to identify the role of β2 ADR in this process.

Ultraviolet radiation is considered a major risk factor for squamous cell carcinoma of the skin^8–10. UVB component of ultraviolet radiation is a complete carcinogen capable of cancer initiation, promotion, and progression⁹. It is therefore clinically relevant in the etiology of squamous cell carcinoma of the skin^11–13. Accordingly, the role of β₂ADR and their selective antagonists on preventing the progression of UVB induced squamous cell tumorigenesis of the skin in SKH-1 hairless mice, a standard animal model used for these studies, have been investigated.

2. MATERIALS AND METHODS

2.1. Cell Culture and Reagents

Mycoplasma free keratinocyte derived VEGF secreting A-431 cells¹⁴ (Cat#CRL-1555, ATCC, MD, USA) was authenticated using short tandem repeat profiling and cultured in Dulbecco’s Modified Eagle’s Medium (Cat#30–2002, ATCC) supplemented with 10% FBS (Cat#30–2020, ATCC). The cells were then serum and growth factor starved for 12 h and treated with selective β₂ ADR antagonists butoxamine (Cat#B1385, Sigma) or ICI-118,551 (ICI) (Cat#0821, Tocris, MN, USA). Forskolin was procured from Sigma, MO, USA.

2.2. UVB Irradiation and Treatment

The animal experiments were approved by the institutional animal care and use committee of the Ohio State University. 6–8 week-old male Skh-1 mice (Charles River Laboratories, CA, USA) were dorsally exposed to UVB radiation (290–320 nm) thrice a week for 16 weeks at a consistent dose of 2240 J/m², using Phillips FS40UVB lamps (American Ultraviolet Company, IN, USA) fitted with Kodacel filters (Eastman Kodak, NY, USA). The UVB dose was determined by a UVX radiometer (UVB, Inc., CA, USA)¹². Following UVB irradiation, the randomly grouped animals were treated with selective β₂ADR antagonists, butoxamine or ICI at a dose of 10 mg/kg/d i.p.^{15, 16} for 14 consecutive days. Skin tumors greater than 1 mm were counted and measured in each mouse using a caliper before the first day of treatment and thereafter, every alternate day throughout the treatment. Finally, at the end of the study, the animals were euthanized, and multiple dorsal skin lesions and tumors were harvested and fixed in 10% neutral buffered formalin^{12, 17}.

2.3. Immunohistochemistry

Skin tissue sections were first stained with anti-CD31 antibody (Cat#ab28364, Abcam, MA, USA) or anti-VEGFA antibody (Cat#ab46154, Abcam) and kept overnight at 4°C and then incubated with secondary antibody (Vector, CA, USA). Images were obtained using Axio Scope upright light microscope (Carl Zeiss, Oberkochen, Germany). Microvessel density, i.e., CD31 staining, a marker of angiogenesis, was enumerated from 10 random fields per section using ImageJ software (National Institute of Health, MD, USA)¹⁸.

2.4. Immunofluorescence

Mouse skin sections were probed with two unconjugated primary antibodies anti-pan keratin antibody (Cat#4545, Cell Signaling Technology, MA, USA) and anti-β₂ ADR antibody (Cat#ab182136, Abcam) overnight at 4°C and then incubated with fluorochrome-conjugated secondary antibodies (Invitrogen, CA, USA) for 1h at room temperature. 4, 6-diamidino-2-phenylindole (DAPI) from Electron Microscopy Sciences, PA, USA, was used as a nucleus counterstain. Slides were visualized using a confocal scanning microscope (FBV-1000; Olympus Corporation, Center Valley, PA, USA)^{19, 20}.

2.5. Enzyme-Linked Immunosorbent Assay

A-431 cells were serum-starved for 12 h and then treated with 1 μM of Butoxamine or ICI for 48 h, and the supernatant was collected. Enzyme-linked immunosorbent assay (ELISA) was done using a Quantikine Enzyme-linked immunosorbent assay kit (Cat#DVE00, R&D Systems) following the protocol of the manufacturer to determine VEGF concentrations in the supernatant. The absorbance was measured at 450 nm, using a PowerWave XS Microplate reader (BioTek Instruments, USA)^{21, 22}.

2.6. Tumor grading

Tumor tissue sections were graded in a blinded manner by board-certified pathologists. The area of the tumor was measured by multiplying the length and width. Tumor burden per mouse was counted from the total tumor area per mouse, which was the sum of all tumor areas from each mouse^{12, 17, 23, 24}.

2.7. Western Blot

A-431 cell lysates were harvested and used for the western blot to assess phospho-cyclic AMP responsive element-binding protein (p-CREB) protein expression (Cat#9198, Cell Signaling Technology, USA). The immunoblot signals were developed using SuperSignal chemiluminescent substrates (Thermo Scientific, USA, Cat# 34580) and visualized using the C-DiGit® Blot Scanner (LI-COR Biosciences, USA, Model# 3600). Protein loading was normalized using an anti-GAPDH antibody (Cat#sc-25778, Santa Cruz Biotechnology). The antibody reactive bands were then quantified through densitometry²⁵.

2.8. Statistics

Data are expressed as mean ± SEM values. Student’s t-test and ANOVA were performed to analyze results from independent groups. Repeated measures ANOVA was done to analyze data obtained on days 0–14. p<0.05 was considered statistically significant. Bonferroni correction was performed for multiple comparisons²⁵.

3. RESULTS

3.1. VEGF and CD31 expressions in UVB-irradiated skin lesions

VEGF is a potent stimulator of angiogenesis^{26, 27}. Therefore, we assessed VEGF and CD31 expressions in UVB irradiated murine skin lesions. The immunohistochemistry results revealed significant upregulation of VEGF and CD31expressions in these lesions compared to normal skin controls (FIGURES 1A–C) (*P < 0.05).

FIGURE 1.

(A) Immunohistochemistry staining of VEGF in normal mouse skin and UVB induced premalignant mouse skin lesions. The arrows show the VEGF expression. N=10 for both groups. (B and C) Immunohistochemistry staining of CD31, i.e., microvessel density in normal mouse skin and UVB induced premalignant mouse skin lesions. The arrows show microvessels. Data are expressed as mean ± SEM. N=10 for both groups. *p < 0.05 versus normal skin, Student’s t test. Scale bar = 100 μm. The figure is representative of ten separate experiments with similar results.

3.2. β₂ ADR are present on keratinocytes in UVB-induced skin lesions

We next determined the status of β2 adrenergic receptors on the keratinocytes in UVB-induced cutaneous lesions in mice. Immunofluorescence staining revealed colocalization of β₂ adrenergic receptors with pan-keratin, a marker of keratinocytes, thereby indicating expression of β₂ adrenergic receptors in the keratinocytes of these lesions (FIGURE 2A).

FIGURE 2.

(A) Immunofluorescence staining and confocal microscopy were used to colocalize pan keratin (green) and β₂AR (red) in mouse premalignant skin lesions. N= 10. Scale bar = 100 μm. The arrows show co-staining. (B and C) Average tumor numbers were recorded and compared between mice treated with selective Butoxamine or ICI and control mice treated with vehicle. Data are expressed as mean ± SEM. N=10 for all groups. *p< 0.05 versus vehicle-treated control, ANOVA with repeated measures. (D and E) Average tumor burdens were recorded and compared between mice treated with Butoxamine or ICI and control mice treated with vehicle. Data are expressed as mean ± SEM. N=10 for all groups. *p< 0.05 versus vehicle-treated control, ANOVA with repeated measures. (F) Gross pictures of the dorsal skin of ultraviolet B irradiated Skh-1 mice treated with Butoxamine or ICI or vehicle. N=10 for all groups. The figures are representative of ten separate experiments with similar results.

3.3. Treatment with β₂ ADR antagonists decrease the number of tumors, the tumor burden, and the development of malignant skin tumors

The number of skin tumors and the tumor burden significantly decreased in UVB-irradiated mice following treatment with selective β₂ ADR antagonists butoxamine or ICI (10 mg/kg once daily i.p. for 14 d) in comparison to vehicle-treated controls (FIGURES 2B–F) (*P < 0.05). On histopathological examination, the lesions were hyperplasia and papilloma (grades 1–3). Although 16.3% of skin lesions were squamous cell carcinomas in the vehicle-treated control group, malignant squamous cell carcinoma was not found in the groups treated with selective β₂ ADR antagonists.

3.4. β₂ ADR antagonists inhibit angiogenesis

Since β₂ ADR are expressed in the keratinocytes in UVB-irradiated skin lesions (FIGURE 2A), we examined the effects of inhibition of β₂ADR actions in these cells by β₂ ADR antagonists on VEGF induced angiogenesis. Mice irradiated with UVB thrice a week with consistent dosing of 2240 J/m² for 16 weeks were treated intraperitoneally (i.p.) on week 17 with selective β₂ ADR antagonists/blockers butoxamine or ICI at a dose of 10 mg/kg once daily for 14 consecutive days. After the end of treatment, i.e., on day 14, our results showed significant inhibition of CD31 and VEGF expressions in the skin lesions of UVB irradiated animals in comparison to vehicle-treated controls (FIGURES 3A–C) (*P < 0.05).

FIGURE 3.

(A, B) Immunohistochemistry staining of CD31, i.e., microvessel density (MVD) in premalignant skin lesions from vehicle-treated control and β₂AR antagonists treated mice. The arrows show the microvessels. Data are expressed as mean ± SEM. N= 10 for both groups. *p < 0.05 versus vehicle-treated control, Student’s t test. Scale bar = 100 μm. The figures are representative of ten separate experiments with similar results. (C) Immunohistochemistry staining of VEGF in premalignant skin lesions from β₂AR antagonists Butoxamine or ICI treated mice compared to vehicle-treated control. The arrows show VEGF expression. N= 10 for all groups. Scale bar = 100 μm. The figures are representative of ten separate experiments with similar results. (D) VEGF concentration in the A-431 cell culture supernatant detected by Enzyme-linked immunosorbent assay. Data are expressed as mean ± SEM. *p < 0.05 versus untreated control, Student’s t test. The figures is representative of six separate experiments under similar conditions. (E) Western blot of A-431 cells treated with Butoxamine or ICI. Forskolin is the positive protein kinase A control. The blots were first probed with phospho-CREB antibody and re-probed with GAPDH antibody to compare equal protein load. The pCREB signals were normalized using the GAPDH antibody. *p < 0.05 versus untreated control, ⁺p < 0.05 versus untreated control (one-way ANOVA). The molecular weight of pCREB is 43 kDa. The figures are representative of six separate experiments under similar conditions.

3.5. Inhibition of β₂ ADR actions in keratinocytes suppress VEGF synthesis

Suppression of VEGF synthesis through inhibition of β₂ ADR actions in the keratinocytes was further confirmed by enzyme-linked immunosorbent assay, which showed significant inhibition of VEGF concentration in the cell culture supernatant of β₂ ADR expressing A-431 cells^{28, 29} upon treatment with 1 μM of selective β₂ ADR blockers butoxamine or ICI for 48 h (FIGURE 3D) (*P < 0.05).

3.6. Inhibition of β₂ ADR function in keratinocytes suppresses VEGF production through the β₂ADR-CREB Pathway

Inhibition of β₂ADR actions in the keratinocytes downregulates VEGF expressions in these cells (FIGURE 3C, D) and subsequent angiogenesis in UVB-irradiated premalignant skin lesions (FIGURES 3A, B). As β₂ADR classically act through the protein kinase A signaling pathway³⁰, and stimulation of the PKA pathway upregulates VEGF synthesis through the phosphorylation or activation of the cAMP response element-binding protein (CREB) transcription factor³¹, therefore, we investigated the effects of treatment with 1 μM of selective β₂ADR butoxamine or ICI on CREB phosphorylation (phospho-CREB) in A-431 cells. Western blot analysis revealed significant inhibition of phospho-CREB in butoxamine- or ICI-treated A-431 cells compared to the untreated controls (FIGURE 3E) (*P < 0.05). Protein kinase A stimulator forskolin (10 μM)²² was a positive control (FIGURE 3E) (⁺P < 0.05) (See Supplemental FIGURE 1 for full blots).

4. DISCUSSION

This study, for the first time, indicated that suppression of actions of β₂ adrenergic receptors by selective β₂ADR antagonists significantly reduced the number of tumors, tumor burden and incidence of malignant squamous cell tumors in UVB-induced premalignant skin lesions through suppression of angiogenesis. These antagonists acted by downregulating VEGF expression in keratinocytes by inhibiting CREB phosphorylation via the protein kinase A/CREB pathway. It is to be noted here that stimulation of β₂ADR by agonists induces cyclic adenosine monophosphate production, which activates protein kinase A³².

Squamous cell carcinoma of the skin is commonly seen globally, and UVB radiation increases the risk of this malignant tumor^8–10. The presently available treatment does not result in a complete cure as the recurrence rate of this cancer is high, impacting the health budget¹⁰. Therefore, there is a need to develop newer and effective therapies to prevent this disease¹⁰. Our results demonstrate a novel role of β₂ADR in regulating the development and progression of UVB-induced premalignant cutaneous lesions and the efficacy of selective β₂ADR antagonists for their treatment. Interestingly, epidemiological studies have reported no increase in squamous cell carcinoma incidence in patients taking beta adrenergic blockers for other disorders³³. These drugs also have a protective effect on melanoma³⁴. Furthermore, as these classes of drugs with manageable side effects are currently being used in the clinics in other diseases, therefore these molecules may be used in the future to inhibit the progression of UVB-induced precancerous lesions for preventing squamous cell carcinoma of the skin.

Abbreviations

β₂ADR

Beta 2 adrenergic receptor

UVB

Ultraviolet B

VEGF

Vascular Endothelial Growth Factor-A

ICI

ICI-118,551

i.p.

intraperitoneally

CREB

cAMP response element-binding protein

ELISA

Enzyme-Linked Immunosorbent Assay

cAMP

cyclic adenosine monophosphate