**4. Discussion**

Microtubule-targeting drugs are extensively used in breast cancer therapy [64]. However, neurotoxicity and the development of resistance represent a clinical problem [65].

Spindle-targeting drugs, such as inhibitors of several kinases involved in the formation and function of the mitotic spindle, are in development to improve anti-mitotic chemotherapy [66]. PLK1 plays a crucial role in cell proliferation through its effects on chromosome segregation, spindle assembly, maturation of the centrosome, and cytokinesis during mitosis [27]. PLK1 is highly expressed in several human cancers with poor clinical outcome [67–69]. However, if PLK1 overexpression contributes to tumour formation, by inducing mitotic alteration and chromosomal instability, is still a matter of debate [70]. Beyond these conflicting results, PLK1 is currently studied as worthwhile therapeutic target [71]. Notably, the resistance to taxane therapy [71] or to T-DM1 treatment have been linked to PLK1 overexpression in HER2+ BC [72]. However, preclinical success with PLK1 inhibitors, has not translated well into clinical success. Dose-limiting toxicities of PLK1 inhibitors and specificity are critical issues [71].

In accordance with previously reported data, demonstrating TCTP's role as a key mitotic target of Plk1 in regulating anaphase progression [30] we recently, showed that TCTP is a direct substrate of PLK1 in mammary carcinoma cell lines. We also showed that DHA, by targeting phospho-TCTP, induces apoptosis and enhances the efficacy of chemotherapy and trastuzumab in HER2 overexpressing tumour cells [10].

The role of phospho-TCTP in cancer cells has not been thoroughly investigated, and only few studies in the literature show that the phosphorylation of TCTP is required for metaphase-anaphase transition [30,33,73]. In addition, high levels of phospho-TCTP are associated with adverse prognostic factors in breast cancer patients and in neuroblastoma patients, as previously reported by our group [10] and by Ramani et al [74], respectively. Moreover, we also showed that an increase of the nuclear phospho-TCTP level is associated with a poor clinical response to trastuzumab therapy in HER2-positive breast cancer [10].

In this study, we show that the reduction of phospho-TCTP levels induced by DHA produces a phenotype resembling almost that observed following PLK1 inhibition. As cellular models for our studies, we chose the HCC1954 and the HCC1569 cell lines, which resemble HER2 overexpressing tumours with *PI3KCA* mutation and loss of PTEN, respectively. In addition, both cells lines were resistant to trastuzumab therapy. Interestingly, malignant progression of HER2-positive breast cancer is often characterized by aberrant PI3K/AKT activation [1,75,76]. For instance, *PI3KCA* mutations and a high proliferation rate are unfavourable prognostic factors in relapsed and de novo metastatic HER2-positive breast cancers treated with trastuzumab [77]. In addition, the loss of at least one copy of the *PTEN* gene is associated with a poor worse outcome in HER2-positive breast cancer, although it is not ye<sup>t</sup> clear whether it is predictive of trastuzumab resistance [78,79].

The levels of phospho-TCTP are critical during mitotic process. Indeed, MCF10A cells overexpressing the non-phosphorylatable form of TCTP showed numerous anomalies of the mitotic spindle. On the contrary, overexpression of the phosphorylatable form of protected MCF10A cells from aberrant mitosis. These data sugges<sup>t</sup> that the reduction of phospho-TCTP levels could be deleterious for achieving proper cell division in growing conditions (Figure 3). In MCF10 cells overexpressing non-phosphorylatable form of TCTP we found high levels of aberrant mitosis. Abnormal mitosis in these cells occurred without any acquisition of DNA damage (Supplementary Figure S2), which in turn may help these cells to bypass surveillance mechanisms. In this context, we can speculate that overexpression of TCTP may protect from oxidative stress that could induce DNA damage in these cells, in accordance with our previously reported data that show TCTP as critical survival factor against oxidative stress [13]. It has been reported that in parasite *Brugia malayi*, TCTP protects DNA from oxidative damage [80]. However, we cannot exclude that DNA damage could occur during mitosis but it is likely to be transient or below a critical threshold necessary to activate p53 to levels that prevent proliferation. These findings have an important clinical value, as aggressive HER2 + BC cells with mutated p53 could be highly sensitive to the e ffect of DHA.

Remarkably, in line with data from preclinical and clinical studies showing that DHA has good safety profile [39,41,42,51], we also showed that the EC50 of DHA was significantly lower in cancer cell lines (<10 μM) than in non-tumourigenic epithelial cells MCF10A (>30 μM) (Table 1). This suggests that DHA has little side e ffects, thus representing a potentially interesting approach for breast cancer therapy.

DHA is a pro-oxidant agen<sup>t</sup> [53] that increases the cellular levels of ROS and leads to DNA damage, in line with the recent observation that artesunate, whose active metabolite is DHA, induces oxidative DNA lesions and DNA double-strand breaks (DSB) [81,82] (Figure 2). Altogether, these findings have some important implications. First, the reduction of TCTP could contribute to DHA-mediated oxidative damage through the increase of oxidative stress, in line with our previously reported findings [13]. The second is that a critical threshold of DSBs is necessary to prevent mitotic entry [83]. In this context, we can speculate that, in HCC1954 cells, DHA induces DNA damage at an extent below this critical threshold, Therefore, cells can still enter mitosis; thus, contributing to increase genome instability. However, excessive genomic instability has a deleterious e ffect to the viability of cancer cells, and could be explored for therapeutic purposes. Moreover, the increase of ROS levels in *PTEN*-deficient HCC1569 cells or *PIK3CA* mutant-HCC1954 cells could be their Achilles' heel, since it has been reported that high levels of active AKT could increase the susceptibility of cancer cells to oxidative stress [84]. PI3K signalling plays a critical role in cell proliferation, mainly through phosphorylation of AKT. In this context, we can speculate that DHA induces metabolic perturbation through the reduction of AKT phosphorylation levels (Figure 2). This e ffect is further enhanced by T-DM1 (Figure 5). Beyond the induction of severe mitotic perturbations, the inhibition of pAKT by the treatment may contribute to the successful outcomes of DHA and T-DM1 therapy. These data may have clinical relevance since molecular alterations involving the PI3K/AKT pathway are frequently observed in advanced HER2+ BC.

By reducing phospho-TCTP levels, DHA induces both mitotic aberration and the formation of disorganized microtubule structures (Figure 1; Figure 3). Therefore, DHA exacerbated the cytotoxicity of T-DM1 as inhibitor of microtubules. Moreover, analysis of the dose reduction index (DRI) for each drug in their combination indicated that addition of DHA to T-DM1 allowed a dose-reduction for T-DM1 in both cell lines. These data have important clinical implication, as it has been reported that patients treated with T-DM1 have more adverse events than those treated with adjuvant trastuzumab [6] (Table 2).

The combination treatment was more e ffective in killing breast cancer cells when compared to the effect induced by any single agent. Notably, the in vivo data show that the growth of tumour cells resumed after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment improved the long-term e fficacy of T-DM1 and induced a severe and irreversible cytotoxic effect, even after treatment interruption (Figure 4; Figure 6).

Collectively, these results sugges<sup>t</sup> that DHA synergizes with T-DM1 in trastuzumab-resistant cell lines. By reducing phospho-TCTP levels, DHA enhances the e ffects of T-DM1 by increasing its effectiveness as a poison for microtubules, and in turn, may cause a dramatic inhibition of cell growth in aggressive breast cancer resistant to trastuzumab therapy. This new therapeutic protocol could lead to a clinically significant improvement in the response to T-DM1 therapy.
