Head and neck and ovarian cancers are known to have heterogeneous clinical responses to the chemotherapeutic agent cisplatin [1
]. A better understanding of this variable response as well as the development of novel strategies to sensitize resistant tumors to cisplatin would be of great importance for the clinical management of this disease. We have previously shown that the variability of the cisplatin response of a subset of head and neck cancer cell lines is linked to the functional status of the FA/BRCA pathway [6
]. In this study we show that the HDAC inhibitor phenylbutyrate sensitizes cisplatin-resistant head and neck cancer cell lines to cisplatin. This sensitization appeared to be due to the abrogation of the FA/BRCA pathway by phenylbutyrate as well as through a FA/BRCA1-independent mechanism. Specifically, we show that phenylbutyrate inhibits the formation of FANCD2 nuclear foci after cisplatin treatment and this inhibition correlates to a down regulation of the tumor suppressor BRCA1.
There is currently great interest in HDAC inhibitors as anti-cancer agents [25
]. The mechanisms for the anti-tumor activities of HDAC inhibitors include induction of apoptosis, cell cycle arrest, cell differentiation, and abrogation of tumor angiogenesis and invasion [26
]. We have previously shown that the HDAC inhibitor phenylbutyrate down-regulates the anti-apoptosis protein Bcl-XL
and the DNA-dependent protein kinase (DNA-PK) involved in double strand break repair and cellular stress signaling [16
]. The consequence of the down-regulation of these proteins should lead to the lowering of the apoptotic threshold and inhibition of double strand break repair and thus phenylbutyrate and other HDAC inhibitors may have sensitizing properties when combined with radiotherapy or chemotherapeutic agents [26
]. The results from this study concur with a sensitizing function of phenylbutyrate when combined with cisplatin. Measuring cell viability/proliferation, apoptosis and clonogenic survival revealed a more than additive effect when combining phenylbutyrate and cisplatin on head and neck cancer cell lines (Fig. ). Thus, phenylbutyrate may be a useful agent to sensitize recurrent cisplatin-resistant head and neck tumors to cisplatin chemotherapy.
We recently showed that a subset of cisplatin-sensitive head and neck cancer cell lines are defective in cisplatin-mediated induction of FANCD2 nuclear foci [6
]. In this study we show that phenylbutyrate abrogated the formation of FANCD2 nuclear foci following cisplatin treatment (Fig. ). The formation of FANCD2 nuclear foci is thought to be essential for the proper processing of interstrand cross links during S-phase [4
] and thus, the abrogation of FANCD2 foci formation by phenylbutyrate pretreatment is probably responsible for the cisplatin-sensitizing effect of phenylbutyrate in these cells. We also show that phenylbutyrate can sensitize FA/BRCA1-deficient head and neck cancer cells suggesting additional target for cisplatin sensitization by phenylbutyrate. It is possible that the cisplatin-sensitizing effect of phenylbutyrate is related to its role in targeting the expression of the apoptosis-antagonist Bcl-XL [16
How does phenylbutyrate interfere with the formation of FANCD2 nuclear foci? A required step in order for FANCD2 proteins to form nuclear foci is that they become monoubiquitylated at the lys561 residue by the FA nuclear complex, consisting of at least eight different FA proteins [4
]. When cisplatin-induced monoubuitylation of FANCD2 was analyzed for the three head and neck cancer cell lines, we did not observe any inhibiting effect of phenylbutyrate (Fig. ). Thus, phenylbutyrate does not appear to interfere with the FA/BRCA pathway by inhibiting the monoubiquitylation of FANCD2. Another requirement for the formation of FANCD2 nuclear foci is that the cells harbor wild-type BRCA1 [23
]. In a previous study we showed that cisplatin-sensitive cell lines having a non-functional FA/BRCA pathway were BRCA1 defective [6
]. BRCA1-deficient cells are known to be hypersensitive to cisplatin [28
] while BRCA1 over-expression has been shown to lead to increased resistance to cisplatin [31
]. In this study we show that phenylbutyrate treatment leads to a down-regulation of BRCA1 in all three head and neck cancer cell lines tested (Fig. ). Thus, the down-regulation of BRCA1 by phenylbutyrate may partially explain the abrogation of cisplatin-induced FANCD2 foci formation and the cellular sensitivity to cisplatin. We also show that phenylbutyrate must target other pathways in addition to the FA/BRCA1 pathway since the FA/BRCA1-defective cell line UM-SSC-14A was effectively sensitized to cisplatin by phenylbutyrate (Fig. ).
Cisplatin is one of the most commonly used chemotherapeutic agents available today for the treatment of various malignancies [1
]. However, its normal tissue toxicities, variable tumor responses and the selection for cisplatin-resistant cancer cells in reoccurring tumors limit the clinical usefulness of cisplatin. Recent efforts have been focused on screening for agents that sensitize tumor cells to cisplatin by inhibiting the FA/BRCA pathway [12
]. One lead compound that interfered with cisplatin-induced FANCD2 monoubiquitylation and sensitized breast and ovarian cancer cells to cisplatin was the natural and relatively non-toxic compound curcumin. Our study identifies the HDAC inhibitor phenylbutyrate as an additional low toxicity agent that sensitizes cancer cells to cisplatin by interfering with the FA/BRCA pathway. Although further studies are needed to in more detail investigate the mechanisms responsible for the phenylbutyrate-induced abrogation of the FA/BRCA pathway, BRCA1 down-regulation and cisplatin-sensitization, our study opens up the possibility that phenylbutyrate could be used to sensitize cisplatin-resistant head and neck tumors in a clinical setting.