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. 2015 Mar 15;5(4):1337-52.
eCollection 2015.

Targeted inhibition of histone deacetylases and hedgehog signaling suppress tumor growth and homologous recombination in aerodigestive cancers

Stephen G Chun  1 , Hyunsil Park  1 , Raj K Pandita  2 , Nobuo Horikoshi  2 , Tej K Pandita  2 , David L Schwartz  1 , John S Yordy  3
Affiliations

Targeted inhibition of histone deacetylases and hedgehog signaling suppress tumor growth and homologous recombination in aerodigestive cancers

Stephen G Chun et al. Am J Cancer Res. .

Abstract

Standard combined modality therapies for aerodigestive tract malignancies have suboptimal outcomes, and targeting cancer-specific molecular pathways in combination with radiation could improve the therapeutic ratio. Dysregulation of epigenetic modulators such as histone deacetylases (HDACs), and developmental morphogens such as the hedgehog (HH) pathway have been implicated in aerodigestive tumor progression and metastasis. We hypothesized that simultaneous targeting of HDACs and the HH-pathway mediator Smoothened (Smo) represents an opportunity to overcome therapeutic resistance in these cancers. We evaluated the effects of the HDAC inhibitor SAHA and Smo inhibitor GDC-0449 with radiation in multiple aerodigestive cancer cell lines. Isobologram analyses showed that SAHA and GDC-0449 synergistically suppressed cancer cell proliferation in vitro. SAHA and GDC-0449 cooperatively enhanced G0/G1 cell cycle arrest which was associated with up-regulation of p21(waf). GDC-0449 prevented SAHA-induced up-regulation of Gli-1 and Gli-2. Both Smo and Ptc-1 expression was cooperatively suppressed by SAHA and GDC-0449. The combination of SAHA and GDC-0449 induced radiation sensitization with 2 Gy as determined by colony formation assays and cytogenetic analyses, which correlated with higher residual γ-H2AX and 53BP1 foci. In mouse tumor xenografts of the SqCC/Y1 cell line, SAHA and GDC-0449 delayed tumor growth longer and prolonged survival more than either agent alone. In summary, we have identified synergistic effect of HDAC and HH signaling for radiosensitization to improve therapeutic outcomes for aerodigestive malignancies.

Keywords: Histone deacetylases; head and neck cancer; hedgehog; homologous recombination; lung cancer; lysine deacetylases; radiation sensitization; smoothened.

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Figures

Figure 1
Figure 1
Isobologram analysis showing synergistic suppression on cellular proliferation by combined targeting of HDACs and HH signaling. A. Characteristics of aerodigestive tumor cell lines and dose responses to SAHA and GDC-0449. B. Proliferation with SAHA 1 µM and GDC-0449 1 µM. *P < 0.05.
Figure 2
Figure 2
Enhanced G0/G1 cell cycle arrest with SAHA and GDC-0449. A. Effect of SAHA and GDC-0449 on cell cycle distribution in SqCC/Y1 and H1299. B. Representative flow cytometry histograms of DNA content for H1299. C. Immunoblot analysis of histone H3 acetylation status and p21 expression.
Figure 3
Figure 3
Hedgehog pathway modulation in response to HDAC and Smo inhibition. A. Immunoblot analysis of HH pathway gene Ptc-1 expression. B. Expression of HH pathway genes Gli-1, Gli-2, and Smo by RT-PCR. White bar, DMSO; dark grey bar, SAHA; black bar, GDC-0449; light grey bar, SAHA + GDC-0449.
Figure 4
Figure 4
Radiation sensitization by SAHA and GDC-0449. A. SAHA and GDC-0449 had a cooperative anti-proliferative effect and enhanced radiation responses in colony formation assays. B. Expression of the DNA damage marker phospho-γ-H2AX in response to 2 Gy with control, SAHA, GDC-0449, and SAHA + GDC-0449. C. SAHA and GDC-0449 cooperatively suppressed homologous recombination (HR) in combination in an HR reporter assay. *P < 0.05.
Figure 5
Figure 5
Mechanisms of radiation sensitization by SAHA and GDC-0449. A. SAHA and GDC-0449 enhance formation of phospho-γ-H2AX and 53BP1 with 2 Gy of radiation. B. Immunofluorescence of representative cells with DNA damage foci phospho-γ-H2AX and 53BP1. C. Expression of the HR recombinase Rad51 in response to a dose titration of SAHA or GDC-0449. D, the combination of SAHA and GDC-0449 cooperatively down-regulates Rad51 expression. *P < 0.05.
Figure 6
Figure 6
Enhanced formation of radiation induced chromosomal aberrations by SAHA and GDC-0449 in H1299 cell line. A. Representative chromosome aberrations induced by combining SAHA, GDC-0449, and SAHA + GDC-0449 with 1 Gy IR. B. The combination of SAHA + GDC-0449 significantly increased formation of chromosomal aberrations in comparison to all other treatment groups. Arrows, chromosomal aberrations. *P < 0.05.
Figure 7
Figure 7
In vivo effect of SAHA and GDC-0449 on the SqCC/Y1 cell line. A. Tumor growth delay with SAHA, GDC-0449, and SAHA + GDC-0449. B. Mouse survival associated with single and combined treatment of SAHA and GDC-0449.
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