**1. Introduction**

HER2-positive breast cancer (HER2 + BC), which represents about 25–30% of breast cancers, is characterized by the overexpression of the human epidermal growth factor receptor 2 (HER2/neu), a tyrosine kinase receptor (RTK). Therapies with monoclonal antibodies of high a ffinity specific for the HER2 receptor, or combinations of multiple anti-HER2 antibodies, have led to a significant improvement in therapeutic response; despite this, many patients develop resistance to therapy [1–3].

Trastuzumab emtansine (T-DM1) is an anti-HER2 antibody-drug linked to the anti-mitotic agen<sup>t</sup> emtansine (DM1). T-DM1 treatment provides significant clinical benefit in breast cancer patients previously treated with chemotherapy and HER2-directed therapy, and in patients with HER2-positive early breast cancer who had residual invasive disease after completion of neoadjuvant therapy [4–7]. However, some patients may develop disease progression [8,9]. Identification of novel combination therapies for T-DM1 represents a major challenge to improve treatment e ffectiveness and to delay or prevent acquired resistance to HER2 inhibition.

Recently, we found that high levels of the nuclear phosphorylated form of Translationally Controlled Tumour Protein (phospho-TCTP) in HER2 + BC is associated with adverse prognostic factors and with a poor clinical response to trastuzumab therapy, suggesting a possible application of phospho-TCTP as a new marker for breast cancer [10].

TCTP is a highly conserved protein and it has been implicated in di fferent physiological processes, including cell proliferation, cell shape, and resistance to stress [11–15]. Gene knockout studies have revealed that TCTP-deficient mice and TCTP-deficient mutants of Drosophila die early during embryogenesis, suggesting its implication in cell proliferation [12,16]. TCTP is a critical target in cancer therapy [15,17–19]. Moreover, TCTP has been identified as a critical regulator of the tumour suppressor p53 [14,20]. TCTP has been also described as a positive regulator of epithelial-to-mesenchymal transition [21,22]. Numerous clinical data show that TCTP overexpression is associated with tumour progression and poor clinical outcome in many poorly di fferentiated tumours [20,23–26]. Interestingly, in human breast cancer, high-TCTP status associates with poorly di fferentiated aggressive G3-grade tumours, predicting poor prognosis [20]. Moreover, TCTP may mediate many biological functions through the interaction with proteins involved in relevant function in cancer biology. Among these are: i) polo-like kinase 1 (PLK1), a member of the polo-like family of serine/threonine kinases that plays a crucial role in cell-cycle regulation during mitosis [27]; ii) Y-box-binding protein 1 (YBX1), a transcription and translation regulator protein that increase cancer cell invasiveness and spreading [28]; iii) the myeloid cell leukemia-1 protein (Mcl-1), an anti-apoptotic Bcl-2 family member [29]. Despite numerous reports suggesting important functions of TCTP in the context of tumour biology, its precise role is less clear.

Our previously reported data show that TCTP is a direct substrate of PLK1 in mammary carcinoma cells [10]. These data are in line with earlier published evidence [30–32]. Notably, the residues of Ser46 and Ser64 of TCTP, that are specifically phosphorylated by PLK1 [30], are located in a highly conserved intrinsically disordered loop of the protein, which contains a highly conserved TCTP signature. The phosphorylation event of TCTP by PLK1 may be critical for the activity of the protein, as it has been demonstrated that phospho-TCTP detaches from the spindle at the metaphase-to-anaphase transition [33].

It has been reported that TCTP regulates spindle microtubule dynamics during mitosis and its overexpression may lead to microtubule stabilization and alterations in cell morphology [34]. Conversely, TCTP phosphorylation by PLK1 increases microtubule dynamics by decreasing the microtubule-stabilizing activity of TCTP [30,33]. Consistent with this data, it has been reported that TCTP phosphorylating activity is low throughout the cell cycle, but increases in mitosis [11].

All together, these data sugges<sup>t</sup> that TCTP may be a crucial player in mitotic processes and a fine equilibrium between the phosphorylated and non-phosphorylated forms of TCTP is required for maintaining the dynamic state of microtubules.

TCTP is a target of dihydroartemisinin (DHA) [10,35–37]. DHA, the active metabolite of Artemisinin, was discovered as an anti-malarial agen<sup>t</sup> by Dr. Youyou Tu, who was awarded the 2015 Nobel Prize in Physiology or Medicine [38]. Notably, it has been reported that microarray-based mRNA expression of human TCTP is correlated with sensitivity to artesunate (a derivative of artemisinin) in tumour cells, suggesting that human TCTP contributes to the response of tumour cells to the drug [36]. Today, phase 1 clinical trials are underway showing that DHA has good safety and tolerability profile after long-term administration in patients with breast cancer or carcinoma of the uterine cervix [39–41]. These data are compatible with those obtained in malarial patients who, on the contrary, followed pharmacological treatments for shorter times [42,43]. All together, these findings shed light on the potential use of DHA as an anti-tumour agen<sup>t</sup> and support the growing interest in this drug compound [44].

We have previously shown in HER2 overexpressing breast cancer cell lines that DHA, by reducing the expression levels of the phosphorylated form of TCTP, enhances the response to treatment with drugs as doxorubicin, cisplatin and trastuzumab.

Here we have investigated, in depth, the response to DHA in combination with T-DM1 in breast cancer cells resistant to trastuzumab therapy. Our data show that the combination treatments caused growth inhibition through the induction of severe mitotic perturbations, which in turn led tumour cells into an unstable state no longer compatible with viability.
