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. 2017 Aug 23;36(1):112.
doi: 10.1186/s13046-017-0583-4.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics

Liwei Lang  1 , Chloe Shay  2 , Xiangdong Zhao  1 , Yong Teng  3   4   5
Affiliations

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics

Liwei Lang et al. J Exp Clin Cancer Res. .

Abstract

Background: Although major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer.

Methods: The drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry.

Results: We show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation.

Conclusion: This study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development.

Keywords: Arf1; Combination treatment; Exo2; Prostate cancer; Ras; Salirasib.

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Conflict of interest statement

Competing interests

The authors declare no competing financial interests.

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University.

Consent for publication

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Exo2 blocks ERK1/2 activity and inhibits proliferation in prostate cancer cells. a DU145 and PC3 cells were treated with 20 μM of the indicated Arf1 inhibitors for 24 h, and cell lysates were collected for Western blot with the indicated antibodies. b, c DU145 and PC3 cells were treated with the indicated concentrations of Exo2 for 24 h (b) or 50 μM Exo2 for the indicated times (c), and cell lysates were collected for Western blot with the indicated antibodies. d Prostate cancer cell lines DU145, PC3, 22Rv1 and LNCaP were treated with the indicated concentrations of Exo2 for 72 h, and cell proliferation was determined by MTS. e DU145 and PC3 cells were treated with the indicated concentrations of Exo2 for 24 h, and GGA3-PBD agarose beads were used to pull down the GTP-bound Arf1 and Western blot was used to determine the indicated protein levels.*p < 0.05; **p < 0.01
Fig. 2
Fig. 2
Salirasib enhances Exo2-induced repression of phospho-ERK1/2 and proliferation in prostate cancer cells. a Various cancer cell lines were treated with the indicated concentrations of salirasib for 72 h, and cell proliferation was determined by MTS assays. b DU145 and PC3 cells were treated with 50 μM salirasib for the indicated times, and cell lysates were collected for Western blot with the indicated antibodies. c DU145, PC3 and LNCaP cells were treated with 50 μM Exo2 and 50 μM salirasib for 24 h, alone or in combination, and cell lysates were collected for Western blot with the indicated antibodies. d DU145 and PC3 cells were treated with 50 μM Exo2 and 50 μM salirasib for 72 h, alone or in combination, and cell proliferation was determined by MTS assays. **p < 0.01
Fig. 3
Fig. 3
Combination of salirasib and Exo2 suppress migration, invasion and apoptosis of prostate cancer cells more efficiently than either drug alone. a DU145 and PC3 cells were treated with 50 μM Exo2 and 50 μM salirasib for 16 h, alone or in combination, and cell migration was determined by gap closure. b–d DU145 and PC3 cells were treated with 50 μM Exo2 and 50 μM salirasib for 24 h, alone or in combination. Cell invasion was determined by Boyden chamber (b), and cell apoptosis was determined by FITC Annexin V Apoptosis Detection Kit I (c). For quantification of invasion, the matrigel membranes that contained invading cells were dissolved in 10% acetic acid and read colorimetrically at 590 nm (b). Representative images of apoptosis assays are shown in (c) and quantitative data are shown in (d). e DU145 and PC3 cells were treated with 50 μM Exo2 and 50 μM salirasib for 24 h, alone or in combination, and cell lysates were collected for Western blot with the indicated antibodies. **p < 0.01
Fig. 4
Fig. 4
Exo2 exhibits inhibitory activity of prostate tumor growth in vivo. a, b When PC3-derived xenografts had been established, the SCID mice were randomly divided into four groups for treatment with vehicle or the indicated concentrations of Exo2 (n = 5/group). Tumor growth was measured by tumor volume (a), tumor weight and mouse body weight (minus tumor) at the end of the experiment was calculated (b). c, d The xenografts were removed these mice for Western blot with the indicated antibodies. Representative result of Western blot is shown in (c) and quantitative data are shown in (d). **p < 0.01
Fig. 5
Fig. 5
Combination of Salirasib and Exo2 inhibits prostate tumor growth more efficiently than either treatment alone. a, b When PC3-derived xenografts had been established, the SCID mice were randomly divided into four groups for treatment with vehicle, Exo2, salirasib or the combination of Exo2 and salirasib (n = 5/group). Tumor growth was measured by tumor volume and size every 3–5 days (a), and tumor and mouse body weight (b) was calculated at the end of the experiment. c The xenografts were removed from these mice for Western blot with the indicated antibodies. Representative result of Western blot is shown in the left panel and quantitative data are shown in the right panel. d–f The xenografts removed from the drug-treated tumor-bearing mice were processed for IHC with the indicated antibodies. Representative images of IHC are shown in (d, e) and quantitative data are shown in (f). **p < 0.01
Fig. 6
Fig. 6
Schematic representation of the mechanism of combination-mediated inhibition of prostate tumor growth
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