Research Papers:

Valproic acid potentiates the anticancer activity of capecitabine in vitro and in vivo in breast cancer models via induction of thymidine phosphorylase expression

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Oncotarget. 2016; 7:7715-7731. https://doi.org/10.18632/oncotarget.6802

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Manuela Terranova-Barberio, Maria Serena Roca, Andrea Ilaria Zotti, Alessandra Leone, Francesca Bruzzese, Carlo Vitagliano, Giosuè Scogliamiglio, Domenico Russo, Giovanni D’Angelo, Renato Franco, Alfredo Budillon _ and Elena Di Gennaro


Manuela Terranova-Barberio1, Maria Serena Roca1, Andrea Ilaria Zotti1, Alessandra Leone1, Francesca Bruzzese1, Carlo Vitagliano1, Giosuè Scogliamiglio2, Domenico Russo3, Giovanni D’Angelo3, Renato Franco2, Alfredo Budillon1, Elena Di Gennaro1

1Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy

2Pathology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy

3Institute of Protein Biochemistry, National Research Council, Naples, Italy

Correspondence to:

Alfredo Budillon, e-mail: a.budillon@istitutotumori.na.it

Keywords: HDAC inhibitor, valproic acid, thymidine phosphorylase, breast cancer, capecitabine

Received: July 27, 2015    Accepted: December 23, 2015    Published: December 31, 2015


The prognosis of patients with metastatic breast cancer remains poor, and thus novel therapeutic approaches are needed. Capecitabine, which is commonly used for metastatic breast cancer in different settings, is an inactive prodrug that takes advantage of elevated levels of thymidine phosphorylase (TP), a key enzyme that is required for its conversion to 5-fluororacil, in tumors. We demonstrated that histone deacetylase inhibitors (HDACi), including low anticonvulsant dosage of VPA, induced the dose- and time-dependent up-regulation of TP transcript and protein expression in breast cancer cells, but not in the non-tumorigenic breast MCF-10A cell line. Through the use of siRNA or isoform-specific HDACi, we demonstrated that HDAC3 is the main isoform whose inhibition is involved in the modulation of TP. The combined treatment with capecitabine and HDACi, including valproic acid (VPA), resulted in synergistic/additive antiproliferative and pro-apoptotic effects in breast cancer cells but not in TP-knockout cells, both in vitro and in vivo, highlighting the crucial role of TP in the synergism observed. Overall, this study suggests that the combination of HDACi (e.g., VPA) and capecitabine is an innovative antitumor strategy that warrants further clinical evaluation for the treatment of metastatic breast cancer.


Breast cancer remains one of the most serious and common diseases and is second only to lung cancer as the leading cause of cancer death in women [1]. Although advances in breast cancer treatment have led to the development of novel therapeutics in the last years, metastatic breast cancer is largely an incurable disease.

As a monotherapy, capecitabine, an oral prodrug of 5-fluorourcacil (5-FU), is one of the mainstays for the treatment of patients with metastatic breast cancer who are ineligible for or who are pretreated with a more intensive anthracycline- and/or taxane-based regimen. Moreover, capecitabine is approved for use in combination with docetaxel for the treatment of breast cancer patients who have received prior therapy with anthracycline, taxane, and trastuzumab. Furthermore, due to its tolerability and efficacy as a single agent and its lack of cross-resistance with other chemotherapeutics, capecitabine may also be considered a preferred partner to be assessed in novel combination regimens [24]. After its absorption as an intact molecule, capecitabine is converted into 5-FU in the gastrointestinal tract through a three-step process. The key step, the conversion of 5′-deoxy-5-fluorouridine (5′-DFUR) into active 5-FU, occurs primarily in the tumor and is catalyzed by thymidine phosphorylase (TP). Subsequently, 5-FU is metabolized into two active cytotoxic metabolites. The first, 5-fluoro-2-deoxyuridine monophosphate (FdUMP), interferes with DNA synthesis through a reduction in thymidine production following the inhibition of thymidylate synthase (TS). The second, 5-fluorouridine triphosphate (FUTP), inhibits RNA and protein synthesis by competing with uridine-triphosphate for incorporation into the RNA strand [2, 5]. Therefore, TP represents the rate-limiting enzyme in the activation of 5′-DFUR and capecitabine, and its elevated level in many tumors allows for high local concentrations of the active drug [2]. Indeed, in breast cancer, TP has been considered a predictive marker for clinical response to capecitabine [6, 7], which suggests that an increase in TP expression might enhance sensitivity of tumor cells to this prodrug. Conversely, the overexpression of TS has been associated with aggressive breast cancer phenotypes and a worse prognosis, particularly in patients with breast cancer who are treated with 5-FU-based chemotherapy [8].

Through the regulation of acetylation of histone and non-histone proteins, histone deacetylases (HDACs) control cellular functions such as the cell cycle, proliferation, survival, DNA repair and differentiation. Their expression is frequently altered in hematologic and solid tumors [9]. Class-I HDACs (1–3, 8) are predominantly expressed in the nucleus and are the major mediators of histone deacetylation, whereas class-IIa HDACs (4, 5, 7, 9) and class-IIb HDACs (6 and 10) either shuttle between the nucleus and the cytoplasm or are predominantly expressed in the cytoplasm where they function to deacetylate non-histone proteins [9]. A large number of histone deacetylase inhibitors (HDACi) are currently in clinical development as anticancer agents, and three (vorinostat, romidepsin and belinostat1) have been approved by the FDA for the treatment of cutaneous T-cell lymphoma [1012]. Moreover, panobinostat was the first HDACi approved as a combination therapy to treat recurrent multiple myeloma [13]2. In solid tumors including breast cancer, HDACi have failed to show considerable antitumor activity as single agents and are more active in combination with radiotherapy, chemotherapy or other biologicals [14]. Recently, based on the results of a phase-II randomized trial, the HDACi entinostat has been designated as a breakthrough therapy for the treatment of recurrent/metastatic estrogen receptor-positive breast cancer when given in combination with exemestane in postmenopausal women who progressed following non-steroidal aromatase inhibitor therapy [15].

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