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. 2012 Jan 3;106(1):107-15.
doi: 10.1038/bjc.2011.495. Epub 2011 Nov 24.

Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma

R B Erlich  1 , Z Kherrouche, D Rickwood, L Endo-Munoz, S Cameron, A Dahler, M Hazar-Rethinam, L M de Long, K Wooley, A Guminski, N A Saunders
Affiliations

Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma

R B Erlich et al. Br J Cancer. .

Abstract

Background: We examine the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC).

Methods: Head and neck squamous cell carcinoma cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were then determined in vitro and in vivo.

Results: Phosphatidylinositol-3-phosphate kinase, AKT and dual PI3K-mTOR inhibitors caused marked in vitro enhancement of cytotoxicity induced by HDACIs in HNSCC cancer cells. This effect correlates with AKT inhibition and is attenuated by expression of constitutively active AKT. Histone deacetylase inhibitor and phosphatidylinositol-3-phosphate kinase inhibitors (PI3KIs) inhibited tumour growth in xenograft models of HNSCC. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining, respectively. However, we saw no evidence of improved efficacy with an HDACI/PI3KI combination.

Interpretation: That PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against HNSCC in vivo.

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Figures

Figure 1
Figure 1
Vorinostat (vo) induces SCC cancer selective cytotoxicity. SCC cell lines (SCC25, Cal27, SCC9) and HKs were treated with varying concentrations of vofor 24 h. (A) BrdU incorporation and (B) cytotoxicity for three different HNSCC cell lines and HKs were determined as described in the text. Values are means±s.e. of two independent experiments performed in triplicate. (C, D) SCC25 cells were treated with vo (5 μM) for 24 h and the LDH release (C) or PI staining (D) calculated as a percent of total. ***Indicates P⩽0.001 vs control (CTR). Values are mean±s.e.m. of at least three experiments performed in triplicate.
Figure 2
Figure 2
PI3K-AKT inhibitors enhancement of vorinostat (vo)-induced cytotoxicity is mediated by strong and durable AKT inhibition. (A) LDH viability assay for SCC25 cells treated with vehicle (CTR) or a maximal cytocidal dose of vo (5 μM) alone or in combination with LY (10 μM) or uo (10 μM) for 24 h. (B, C) Western blots show lysates from SCC25 cells treated with vo (5 μM) alone or in combination with LY (10 μM) or uo (10 μM) at distinct time points. (D) LDH viability assay for Cal27 cells treated with vehicle (CTR) or vo (5 μM) alone or in combination with LY (10 μM) or uo (10 μM) for 24 h. (E) LDH viability assay for SCC25 cells treated with valproic acid (VA, 3 mM) or Depsipeptide (Depsip, 5 nM) alone or in combination with LY (10 μM). (F) SCC25 cells were transfected with constitutively active AKT (myr-AKT, black boxes) or its corresponding empty vector (open boxes) and then left untreated (CTR) or were treated for 24 h with vo (5 μM)+LY294002 (10 μM, LY). Cell viability was then estimated by LDH release. (G) Western blot of lysates from SCC25 cells showing the expression of the myr-AKT and endogenous AKT in vector only and myr-AKT transfected cells. Phosphorylation of the AKT target GSK3β (p-GSK3β) are provided to confirm functional AKT activity. Western blot figures are representative of two independent experiments. (H) LDH viability assay for SCC25 cells treated with vo (5 μM) alone or in combination with LY (10 μM) or wortmannin (1 μM). (I) Similar assay for cells treated with vo (5 μM) alone or in combination with AKTVIII (10 μM) for 24 h. LDH values presented as a percent of total LDH. Western blot figures are representative of at least three independent experiments. *Indicates P⩽0.05. **Indicates P⩽0.01. ***Indicates P⩽0.001 vs CTR. Values presented as mean±s.e.m. of at least three independent experiments performed in triplicate.
Figure 3
Figure 3
Clinically relevant PI3K-AKT-mTOR inhibitors enhance cancer cell specific cytotoxicity induced by LBH589. (A) Western blots show lysates from SCC25 cells treated for 48 h with LBH589 (300 nM), BEZ235 (300 nM), BKM120 (300 nM) and BGT226 (300 nM), alone in combinations. (B) Viability assay for SCC25 cells subjected to the same treatments as in (A). (C) Western blots show lysates from normal HKs subjected to the same treatments as in (A). (D) Viability assay for normal HKs cells subjected to the same treatments as in (C). Western blot figures are representative of three independent experiments. Values are means±s.e. of three independent experiments performed in triplicate. *Indicates P<0.05.
Figure 4
Figure 4
Antitumour properties of LBH589 and PI3K/mTOR/AKT inhibitors in a xenotransplant model of HNSCC. (A–C) Six week old NOD/SCID mice (groups of four) were injected with 2.5 × 105 Cal27 cells on day 0. 28 days after injection of cells mice were treated with (i) vehicle only, (ii) LBH589 (30 mg kg −1day−1 i.p.), (iii) BEZ235 (30 mg kg −1day−1 p.o.), (iv) BGT226 (10 mg kg −1day−1 p.o.), (v) BKM120 (7.5 mg kg −1day−1 p.o), (vi) LBH589 (30 mg kg −1day−1 i.p.)+BEZ235 (30 mg kg −1day−1 p.o.), (vii) LBH589 (30 mg kg −1day−1 i.p.)+BGT226 (10 mg kg −1day−1 p.o.), (viii) LBH589 (30 mg kg −1day−1 i.p.)+BKM120 (7.5 mg kg −1day−1 p.o.). Tumour volume was measured over a period of 60 days post-tumour inoculation or until a volume of 1 cm3 was reached. Data presented as mean±s.e.m. of individual measurements from four mice per group. Dotted line indicates beginning of treatment for distinct groups.
Figure 5
Figure 5
Assessment of the effects of PI3K inhibitors and HDACIs in tumour tissue. Representative figures shows immunostaining for (A) p-AKT, (B) acetyl-histone H3 and (C) active caspase 3 (bars=100 μm). (D) Active caspase 3 labelling quantification and (E) BrdU labelling quantification in tumours derived from mice subjected to treatment with BEZ235, BGT226 and BKM120 alone or in combination with LBH589. (F) Viability assay following treatment of the parental SCC cell line Detroit and distinct clonal variants with LBH589 (300 nM), BEZ235 (300 nM) or both for 48 h. (G) Viability assay following treatment of the parental Detroit 562 SCC cell line and distinct clonal variants with BGT226 at 0, 100 or 300 nM for 48 h (H) Viability assay following treatment of the parental Fadu SCC cell line and distinct clonal variants with BGT226 at 0, 100 or 300 nM for 48 h. Values for (D) and (E) are means±s.e. for at least three independently treated mouse tumours. Values for (F) and (G) are means±s.e. from triplicate determinations.
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