Abstract 1876: Combination of PI3KCA targeting with irradiation: A preclinical study on head and neck cancer cell lines
Authors: Lattanzio Laura , Tonissi Federica , Monteverde Martino , Gianello Luca , Russi Elvio , Merlano Marco C. , Nigro Cristiana L. , Milano Gerard 
Published: May 2015

Purpose

In complement to anti-EGFR therapy, the targeting of PI3KCA/AKT/mTOR signalling pathway is of particular interest in the management of HNSCC. Its activation is recognized as an important mechanism in tumor progression and resistance to EGFR inhibitors. Of importance, PI3KCA inhibitors have shown their ability to selectively inhibit the PI3KCA/AKT signalling and to enhance the effects of conventional cytotoxic agents. Therefore, it would be particularly interesting to assess the effects of PI3KCA inhibition combined with anti-EGFR monoclonal antibody (cetuximab) and/or irradiation (RT).

Experimental procedures

We identified two HNSCC cell lines (Cal 33, PIK3CA H1047R mutated and Cal 27, wild type) and examined the effects of the combination of BKM120 (specific inhibitor of PI3KCA), cetuximab and RT. Experimental conditions were: BKM120, cetuximab, RT alone; sequence 1 (BKM120 followed by cetuximab), sequence 2 (cetuximab followed by BKM120) and combination (BKM120 + cetuximab together) with/without RT given ahead of all treatments. Drugs were administered at their IC50. Cell survival was assessed by MTT test. Combination effects were analyzed by the Combination Index (CI) and Compusyn software.

Biochemical factors were examined for the impact on proliferation, apoptosis (caspases), DNA repair (ERCC1, XRCC1, DNA-PK, ATM) and PI3KCA/AKT/mTOR pathway p-P70/S6k, p-Akt/Akt, PTEN). Gene expression was determined by real time-PCR and protein expression by western blot.

Results

We evidenced sequence 2 as the best combined treatment in inhibiting cell proliferation in both cell lines (ΔIC50 = - 7.91*10-6 M in Cal 7 and ΔIC50 = - 5.7*10-6 M in Cal 33; being ΔIC50 the difference of IC50 between sequence 2 and BKM120 alone). CI values confirmed a synergistic interaction between the two drugs given sequentially cetuximab + BKM120 in both cell lines.

Addition of RT ahead of all treatments did not add any anti-proliferative effect to the treatments on Cal 33. Of note, on Cal 27 the addition of RT to BKM120 increased the antiproliferative effect of this drug (p&lt;0.05).

Overall, preliminary data on protein expression indicated an upregulation of caspase 3 and 9 only in Cal 27 suggesting an apoptotic pathway activation after sequence 1 and 2, not seen in Cal 33.

Moreover Akt was upregulated in Cal 27 after sequence 1 and 2. Analysis of pAkt is on going to verify if blocking of PI3KCA by BKM120 in our models could lead to a control of the feedback loop induced by mTORC1 inhibition. This may be supported by the observed upregulation of p4EBP1 (downstream) following sequence 1 and 2. Akt upregulation due to the treatments may also explain the lack of a significative effect of RT on cell proliferation, mainly in Cal 27 wild-type cells.

Conclusions

The results of this in vitro could serve as a basis for early clinical trials associating BKM120 and RT in locally advanced HNSCC, providing interesting biochemical markers to conduct personalised therapy.

Citation Format: Laura Lattanzio, Federica Tonissi, Martino Monteverde, Luca Gianello, Elvio Russi, Marco C. Merlano, Cristiana Lo Nigro, Gerard Milano. Combination of PI3KCA targeting with irradiation: A preclinical study on head and neck cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1876. doi:10.1158/1538-7445.AM2014-1876