**3. Results**

### *3.1. DHA A*ff*ects Mitosis of HER2*+ *BC Cell Lines with Aberrant PI3K*/*AKT Signalling*

We investigated the effect of DHA on HER2+ breast cancer cells resistant to trastuzumab. Since PI3KCA mutations and/or loss of phosphatase and tensin homolog (PTEN) have been associated with a lower response to trastuzumab and chemotherapy [1,48,49], we chose the HCC1954 cell line characterized by a mutation in the catalytic domain (H1047R) of *PI3KCA* and the HCC1569 cell line, which is PTEN-null. Both cell lines showed resistance to trastuzumab or pertuzumab therapy, in line with the data in literature [50], whereas HER2-positive breast cancer BT-474 cell line was sensitive to antibody therapy (Supplementary Figure S1).

Cells were grown in the presence or absence of DHA. As shown in Figure 1 (panel a), DHA induced a mild but significant inhibition of cell viability during the first 24–48 h at concentrations ranging from 1.25 to 5 μM, which are achievable in the clinic [40,42,43,51]. Western blot analysis also showed that DHA at these concentrations induced a reduction of both total and phospho-TCTP levels, in accordance with our previous data (Figure 1b) [10]. PLK1 inhibition impairs TCTP phosphorylation [10,52] as does DHA. Since PLK1 is a master mitotic regulator [27], we investigated the impact of DHA on mitotic cells. To this end, we performed a morphological analysis in both cell lines after 24 h of exposure to DHA. Control mitotic cells were characterized by a bright peri-chromosomal localisation of both phospho-TCTP (Figure 1c, left panel), and TCTP (Figure 1c, right panel), in accordance with data from the literature [31]. The signal intensity of phospho-TCTP was higher around the chromosomes than in the rest of cells, only in untreated cells, as indicated by a quantification analysis of peri-chromosomal localization of phospho-TCTP in both DHA-treated and untreated cells (Figure 1d). No differences in the signal intensity of TCTP were observed between untreated and treated cells (data not shown).

Interestingly, DHA-treated cells showed a significant increase of aberrant spindle structures (Figure 1e), such as multipolar or monopolar spindles, or chromosome misalignments (Figure 1c). The treatment did not arrest the progression of these cells through G2 and mitosis (Figure 1f). However, the cell cycle distribution analysis showed that DHA blocked the G1/S transition of a small, ye<sup>t</sup> remarkable, percentage of HCC1569 cells (Figure 1f, upper panel). In addition, Western blot analysis showed that DHA induced an increase of Cyclin B1 and phospho-Histone-H3 in HCC1954 cells, thus suggesting that a delay in mitotic progression could be induced by treatment (Figure 1b). Altogether, these data show that aberrant mitosis could be induced by an early treatment with DHA in aggressive HER2+ BC cell lines and a reduction of the peri-chromosomal localization of phospho-TCTP level occurs at the same time.

### *3.2. DHA Induces a Decrease in AKT Phosphorylation Levels and DNA Damage Through the Increase of ROS in HER2*+ *BC Cell Lines*

Then we investigated whether DHA could prevent phosphorylation on AKT's activation sites, as it has been demonstrated that active AKT is a crucial player in the downstream HER2 signalling pathways [1]. To this end, we evaluated the level of active AKT, in both cell lines after 24 h of DHA treatment. Western blot analysis of the total cell lysates showed that DHA induced a decrease in AKT phosphorylation levels (Figure 2c). DHA contains an endoperoxide moiety [53] that generated reactive oxygen species (ROS) in the first hours of treatment (Figure 2, panel a). To determine the implication of oxidative stress in the cytotoxicity of DHA, cells were pretreated with the ROS scavenger N-Acetyl-L-Cysteine (NAC) for 3 h followed by treatment with DHA. DHA-induced increase in ROS levels was abrogated by NAC (Figure 2a). In addition, NAC administration partially protected the cells from the anti-proliferative effect of DHA (Figure 2b), thus indicating that DHA cytotoxicity may be mediated at least in part by increase of ROS.

Then we investigated whether the phosphorylation of AKT can also be regulated by ROS. Notably, NAC reversed the inhibition of AKT phosphorylation induced by DHA treatment in both cell lines (Figure 2c). Since the increase of ROS levels may induce oxidative DNA damage in DHA-treated cells, we examined the phosphorylation levels of histone 2AX (γH2AX), as a marker of DNA double-strand breaks and genomic instability, in both cell lines after 24 h of exposure to DHA. A mild increase of H2AX phosphorylation at Ser139 was clearly detectable at 24 h of DHA treatment in both cell lines and it was reverted by NAC (Figure 2c).

These data show that, beyond the induction of mitotic perturbations, through the increase of oxidative stress, DHA could further damage cancer cells and, thus, might render them more sensitive to a subsequent treatment.

**Figure 1.** Dihydroartemisinin (DHA) induces aberrant mitosis in HER2-positive breast cancer (HER2+ BC) cell lines. (**a**) Cell viability was assessed by ATP assay. Data are expressed as the percentage of viable cells relative to controls. Values represent the mean ± SD, n = 3. (**b**) Western Blot analysis of the indicated proteins in cell lysates of cells treated with DHA 24 h. β-actin was used as loading control (right panel). For densitometric analysis, the intensity of each band was normalized to the respective β-actin (left panel). (**c**) Immunofluorescence detection of phospho-Translationally Controlled Tumour Protein (pTCTP S46) (red) or TCTP (red) and αTubulin (green) in cells treated with DHA at 2.5 μM for 24 h. Nuclei were stained with Hoechst (blue). The overlay of the three fluorochromes is shown (Merge), bar = 5 μm. Data from a representative experiment of three with similar results are shown. (**d**) Quantification of p-TCTP. Cells were treated as described in (**c**). The line profile shows the distribution of pTCTP as a function of the distance from the chromosome. The quantification was performed as described in Materials and Methods. (**e**) Bar graphs show relative quantification of fraction of cells with aberrant mitosis. At least 1000 cells were analysed in each group. Values represent the mean ± SD. (**f**) Cell cycle distribution in cells treated with DHA for 24 h. Values represent the mean ± SD, *n* = 3. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001 vs. control.

**Figure 2.** DHA induces oxidative stress in HER2+ BC cell lines. (**a**) Reactive oxygen species (ROS) production was assessed in cells pretreated with N-Acetyl-L- Cysteine (NAC) (10mM) followed by treatment with DHA for 24 h. ROS production was measured at the end of incubation time. Data are expressed as ROS levels relative to controls. n = 5. (**b**) Trypan blue exclusion assay was performed in cells treated as described in a). Data are expressed as the percentage of viable cells relative to controls. Values represent the mean ± SD, n = 5. (**c**) Western Blot analysis of the indicated proteins in HCC1569 (upper panel) and HCC1954 (lower panel) cells treated as described in a). β-actin was used as loading control. For densitometric analysis, the intensity of each band was normalized to the respective β-actin. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.01.

### *3.3. Phosphorylation of TCTP is Required for Correct Mitotic Progression in Human Mammary Cells*

To investigate the role of phospho-TCTP in cells committed to mitosis, we performed a series of experiments using the MCF10A cells, a non-tumorigenic human mammary cell line, which expresses low phospho-TCTP and TCTP levels (as previously demonstrated [10]), in which we overexpressed WT FLAG-tagged TCTP protein (WT) or a non-phosphorylatable mutant (AA) TCTP protein. To this aim, we carried out a retroviral infection to establish stable MCF10A subclones expressing: 1) empty vector, hereafter called MCF10A-pBabe; 2) wild-type TCTP protein (WT-TCTP), hereafter called MCF10A-WTTCTP; 3) Ser46Ala Ser64Ala double mutant TCTP (AA-TCTP), hereafter called MCF10A-AATCTP. As shown in Figure 3a, the expression of both forms (WT-TCTP and AA-TCTP) was verified assessing the level of tagged proteins by Western Blot (Figure 3a, left panel) and immunofluorescence analysis (Figure 3a, right panel).

**Figure 3.** *Cont.*

**Figure 3.** Effects of DHA on mitosis in human mammary cells. (**a**) Western blot analysis of FLAG-tagged TCTP protein in cell lysates of MCF10A-pBabe (pBabe), MCF10A-AATCTP (AA), MCF10A–WTTCTP (WT) cells after 6 days of exposure to DHA. β-actin was used as loading control (upper panel). Immunofluorescence detection of FLAG (green) fusion proteins, bar = 25 μm. (**b**) Cell viability was assessed by ATP assay in cells treated with DHA for 6 days. Data are expressed as the percentage of viable cells relative to controls. Values represent the mean ± SD, n = 3. \* = *p* < 0.05, \*\* = *p* < 0.01. (**c**) Growth curve for all cell clones. (**d**) Immunofluorescence detection of Ki-67 (red). Nuclei were stained with Hoechst (blue). The overlay of the two fluorochromes is shown (Merge). Images are shown as single slice of a projection, bar = 100 μm. Data from a representative experiment of two with similar results are shown (left panel). Quantification of Ki-67 positive cells was performed as described in Materials and Methods (right panel). Values represent the mean ± SEM, \* = *p* < 0.05. (**e**) Immunofluorescence detection of phospho-TCTP (pTCTP S46) (red) and αTubulin (green). Nuclei were stained with Hoechst (blue). The overlay of the three fluorochromes is shown (Merge). Cells were treated with DHA (50 μM) for 6 days. Bar = 5 μm). Data from a representative experiment of three with similar results are shown (left panel). Quantification of fraction of cells with aberrant mitosis. At least 1000 cells were analysed in each group. Values represent the mean ± SD. (right panel). (**f**) Immunofluorescence detection of αTubulin (green) in cells treated as described in (**e**), bar = 25 μm), left panel. Quantitative analysis of the microtubule density. The quantification was performed as described in Materials and Methods. Values represent the mean ± SD, right panel.

We compared MCF10A-pBabe cells to MCF10A-AATCTP and MCF10A-WTTCTP cells for their resistance to DHA. To this end, all cell clones were grown in the absence or presence of different concentrations of DHA for 6 days. As shown in Figure 3b, DHA induced an inhibitory effect in all three cell clones at significantly higher concentration than those active on cancer cells (>10 μM) (Table 1), suggesting that DHA can target cancer cells without harming healthy tissues. Overexpression of TCTP protected cells against DHA induced-cytotoxicity, as indicated by the fold increase in half-maximal effective concentration EC50 values reported in Table 1.

**Table 1.** Effects of DHA on cell growth in human mammary cells and in HER2+ BC cell lines. MCF10A-pBabe, MCF10A-AATCTP and MCF10A-WTTCTP cells were treated as described in legend 3b. HCC1569 and HCC1954 cells were treated as described in legend 4a. Half-maximal effective concentration (EC50) values derived from concentration-response curves to DHA. Values represent the mean ± SD, *n* = 3. Significant differences between EC50 values from breast cancer cell lines and MCF10A cell clones are indicated, \*\*\* = *p* < 0.01.


We also found that overexpression of TCTP conferred a growth advantage. A growth curve analysis clearly demonstrate that doubling time of MCF10A subclones overexpressing both WTTCTP (46.79 h) and AATCTP (47.36 h) was shorter than that of MCF10A pBabe (71.88 h), indicating a more rapid proliferation (Figure 3, panel c). Moreover, quantification immunofluorescence analysis showed that both MCF10A-AATCTP and MCF10A-WTTCTP cells had higher expressions of Ki-67, a marker of cell proliferation, which is not detected in non-cycling cells (Figure 3d).

However, the overexpression of the non-phosphorylatable form of TCTP leads to an increase of a phenotype characterized by mitotic aberration [30]. In line with this data, control MCF10A-AATCTP cells showed aberrant mitotic figures, as shown by an immunofluorescence analysis (Figure 3e, left panel) and by a quantification analysis (Figure 3e, right panel). DHA at a concentration of 50 μM induced in these cells a severe damage, as indicated by a quantitative analysis showing an increase in microtubule mass mainly in MCF10A-AATCTP cells **(**Figure 3f). In contrast, we did not find any relevant alterations to the spindle morphology or to microtubule density between treated and non-treated cells in MCF10A-WTTCTP (Figure 3e,f) suggesting that expression of the phosphorylated form of TCTP is required for correct mitosis. DHA induced an increase in ROS levels in all cell clones. However, the increase in ROS levels after treatment was significantly higher in MCF10A-AATCTP cells when compared to MCF10A-WTTCTP cells (Supplementary Figure S2, panel a). The phosphorylation of AKT was not substantially affected by DHA and only a very light inhibition was observed in AA-MCF10 cells (Supplementary Figure S2, panel b). Moreover, DHA induced a very light increase of γH2AX levels only in MCF10A-AATCTP cells in comparison to the other cell clones (Supplementary Figure S2, panel b), thus indicating a greater vulnerability of these cells to DNA damage.

Altogether, these data show that TCTP is a crucial player in mitotic processes. By reduction of the phosphorylated form of TCTP, by mutagenesis and by DHA treatment, resulted in mitotic aberrations and increased microtubule density.

### *3.4. DHA Enhances T-DM1 <sup>E</sup>*ffi*cacy in Breast Cancer Cells Resistant to Trastuzumab Therapy*

We then investigated the combinatorial effects of DHA and T-DM1 in both HCC1954 and HCC1569 cell lines. In a pilot study, we used two different protocols. In the first protocol, the effect of T-DM1 was studied in cells pre-treated with DHA and then exposed to T-DM1, while in the second protocol the drugs were administered at the same time. However, no remarkable di fferences in term of e fficacy and CI values were obtained between the two protocols in both cell lines (data not shown). Since DHA induced mitotic aberration during the first 24 h of exposure, we decided to follow the first protocol.

HCC1954 cells were sensitive to both T-DM1 and DHA treatments as indicated by parameters from the dose-response curves obtained with various concentrations of these drugs (Table 2). Notably, T-DM1 showed a sharp increase in the slope of a dose-response curve, indicating a high cytotoxicity above the EC50 value.

**Table 2.** DHA in combination with T-DM1 causes synergistic inhibition of growth in HER2+ BC cancer cell lines. Cell viability was assessed by ATP assay in cells treated as described in legend 4a. Fractional inhibition = fraction decreased cell viability after treatment, control cells were set to "1". The parameters m, Dm, and r are the shape of the dose-e ffect curve, the potency (EC50), and the conformity of the data to the mass-action law, respectively. CI values below 0.9 indicate synergistic e ffect. CI values = 1 indicate additive e ffect. DRI (DHA) and DRI (T-DM1) are the dose reduction index for DHA and T-DM1, respectively. Value represent the mean ± SD, n = 3.


Drugs were tested at di fferent concentrations in order to find out which ratio yielded a better response. Based on the values of EC50 obtained from the dose-response curve of each drug, a first study was carried out in HCC1954 cells by mixing the two drugs (DHA and T-DM1) at various ratio from 50:1 to 10:1 (data not shown). The two-drug combination at a ratio of 10:1 yielded synergistic inhibition (Table 2). We have also extend the studies at a non-constant combination ratio (e.g., keep DHA at constant dose and vary the dose of T-DM1). DHA was used at concentration ranging from 1.25 to 5 μM (Table 2). Interestingly, the two-drug combination was more e fficacious in inhibiting cell growth when cells were treated with DHA at 2.5 μM and T-DM1 at 0.25 μg/mL (Table 2, and Figure 4a, left panel).

**Figure 4.** T-DM1 when combined with DHA is more effective in killing cancer cells. (**a**) Cell viability was assessed by ATP assay in cells pre-treated with DHA for 24 h and then treated with T-DM1 for 5 days. Relative survival fraction after treatment, control cells were set to "1". Values represent the mean ± SD, *n* = 3 \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001 (**b**) Colony formation assay. Cells were treated as described in (a). A representative experiment of three with similar results is shown. Bar graphs show quantification analysis. Values represent the mean ± SD, *n* = 3. \*\* = *p* < 0.01, \*\*\* = *p* < 0.001. (**c**) Trypan blue exclusion assay was performed in cells pre-treated with DHA for 24 h and then treated with T-DM1 for 3 days (grey columns). On day 4, cells were washed with fresh media and further incubated for additional 4 days (black column). Data are expressed as the percentage of viable cells relative to controls. Values represent the mean ± SD, *n* = 3. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001. (**d**) Quantification of tumour xenografts growth by bioluminescence imaging. Luciferase-expressing HCC1954 cells were implanted into the mammary gland of CB17SCID mice. After 22 days animals were treated with: (1) vehicle; (2) DHA, administrated intraperitoneally (i.p.) 5 days per week, for 4 weeks, in a single daily dose of 25 mg/kg; (3) T-DM1, intravenously (i.v.), once every twelve days for a total of two injections at doses of 10 mg/kg; (4) the combination of DHA and T-DM1. Bioluminescence was detected in mice 11, 39, 60 and 95 days after tumour inoculation. Mean ± SEM is shown. Photon emission is measured as photons/sec/cm2/steradian (N = 5), *P* < 0.05, Mann Whitney test (left panel). Bioluminescence of a representative mouse at the indicated days after tumour inoculation. The intensity of light emission from the animals is represented in pseudo-colour scaling (right panel). \* = *p* < 0.05.

The HCC1569 cell line was less responsive to T-DM1 treatment when compared to HCC1954 cells, as indicated by the EC50 value (EC50 =101.70 ± 44.8) reported in Table 2. We chose an arbitrary ratio of 1:1 of drug combinations in order to identify the minimum e ffective dose of each drug. Results obtained from drug combination studies are shown in Table 2 and Figure 4a (right panel). Overall, we found that DHA synergized with T-DM1, as indicated by the CI values less than 1 (Table 2). Interestingly, analysis of the dose reduction index (DRI) indicated that addition of DHA to T-DM1 allowed a dose-reduction for T-DM1 in both cell lines (Table 2). For instance, the addition of DHA at 2.5 μM to T-DM1 allowed up to 3-fold and ~10-fold reduction of T-DM1 in HCC1954 cells and HCC1569 cells, respectively.

The inhibition of cell growth induced by the combination of DHA and T-DM1 has been also confirmed by colony assay experiments in both cell lines (Figure 4b).

To further test the e fficacy of the combination of DHA and T-DM1, we performed washout experiments. Cells were cultured at low density (1500 cells/cm2) to maintain them in exponential growth. HCC1954 cells and HCC1569 cells were pre-treated with DHA at 2.5 μM and then treated with T-DM1 at 0.25 μg/mL and 2.5 μg/mL, respectively, for 3 days. This was followed by the removal of the drugs from the media and by a further incubation for additional 4 days. At the end of incubation time, the number of viable cells was determined by trypan blue dye exclusion assay. Figure 4c shows that a severe inhibition of cell growth was induced in both cell lines by the DHA and T-DM1 treatment. This e ffect persisted to a greater extent than those of each single agen<sup>t</sup> upon removal of the drug-containing medium.

We then studied the inhibitory e ffects of DHA, T-DM1 and the two-drug combination in vivo. An orthotopic xenograft model of breast cancer was established by implantation of luciferase-expressing HCC1954 cells in the mouse mammary gland. Tumour cell engraftment and early detection of tumour growth was assessed by BLI analysis. To determine the in vivo antitumour activity of the treatments, pharmacological administration was initiated 22 days after tumour cell inoculation, when tumour bioluminescent emission reached an average value of 8 ×10<sup>9</sup> photons/sec/cm2/steradian (Supplementary Figure S3, panel a). Animals were divided into 4 experimental groups (N = 5 per group). Following treatment, mice were monitored for tumour recurrence. Within the period of follow-up, any single agen<sup>t</sup> or the two-drug combination were well tolerated, with no signs of toxicity and weight loss (Supplementary Figure S3, panel b). As expected, T-DM1 treatment was e ffective in inhibiting tumour growth. However, the growth of the tumours resumed after having achieved a complete response (Figure 4d and Supplementary Figure S3, panel c), in line with data from literature [54,55]. In contrast, the combination DHA and T-DM1 was more e fficient than each single agen<sup>t</sup> on tumour inhibition throughout the observation period (Figure 4d, and Supplementary Figure S3, panel c), consistent with the results shown in Figure 4c.

Altogether, these data sugges<sup>t</sup> that T-DM1 when combined with DHA is more e ffective in killing HER2+ BC cell lines.

### *3.5. The E*ff*ects of Two-Drug Combination on HER2-Mediated Cell Signalling*

It has been reported that T-DM1 inhibits AKT phosphorylation in both trastuzumab-responsive and insensitive cell lines, suggesting that this e ffect could be mediated by the DM1 component of T-DM1 [56,57]. Therefore, we assessed the levels of activation of AKT in HCC1954 and HCC1569 cells pre-treated with DHA for 24 h and then exposed to T-DM1 for three days. Western blot analysis showed a grea<sup>t</sup> reduction of active AKT in cells treated with the combination of two drugs as compared to the e ffect induced by each single agen<sup>t</sup> (Figure 5a). After four days of treatment, phospho-AKT levels were not a ffected by DHA (Figure 5a) and this suggests that only the T-DM1 and DHA combination was e ffective enough to reduce active AKT in the long-term period.

Then we investigated the e ffects of two-drug combination on the activation of p44/p42 MAPK (also called ERK1/2). Western blot analysis showed that the levels of phosphorylated ERK1/2 were barely reduced by the two-drug treatment.

**Figure 5.** Effects of Two-drug combination on Protein kinase B (PKB/AKT hereafter referred as AKT), phospho-AMP-activated protein kinase (AMPK), and human epidermal growth factor receptor 2 (HER2) expression in HER2+ BC cell lines. (**a**) Western Blot analysis of the indicated proteins in cells pre-treated with DHA for 24 h and then treated with T-DM1 for 3 days. β-actin was used as loading control (right panel). For densitometric analysis the intensity of each band was normalized to the respective β-actin Quantification analysis was performed by using ImageJ software (left panel). (**b**) Quantitative flow cytometry (FACS) analysis of membrane HER2 expression. Cells were treated as described in (**a**) Histograms show one representative experiment of two with similar results. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.01.

We also studied the phosphorylation of AMPK at its activation site Thr172. Interestingly, the two-drug treatment induced an increase of pphospho-AMPK, which was well-detectable in HCC1954 cells and, to a lesser degree, in HCC1569 cells when compared to control (Figure 5a). Interestingly, AMPK activation may play a role in mitosis and genomic stability beyond its role in the metabolic stress response [58,59] and could be activated upon T-DM1 or DHA treatment.

One critical point in the anti-HER2 therapy is that patients can experience altered HER2 status [60,61], and loss of HER2 expression could lead to a resistant phenotype. We found that T-DM1 treatment induced a reduction of cells with the highest levels of HER2 and an enrichment of cells with low levels of HER2 in HCC1954 cells, suggesting that T-DM1 eliminated cells expressing the highest HER2 levels, but still are responsive to the treatment, in line with data from the literature [9] (Figure 5b). In contrast, the HER2 status was not affected by T-DM1 in HCC1569 cells, which were less responsive to T-DM1 treatment (Figure 5b). Altogether, these data sugges<sup>t</sup> that the inhibition of phospho-AKT could be mediated by the DM1 component of T-DM1 in HER2+ BC cell lines resistant to trastuzumab. A grea<sup>t</sup>

reduction of active AKT is observed in these cells when treated with the combination of two drugs as compared to the e ffect induced by each single agent.

### *3.6. DHA in Combination with T-DM1 Led to Mitotic Catastrophe*

Since T-DM1 is a microtubule-disrupting agen<sup>t</sup> that may cause cell cycle arrest in the G2 and M phases, we evaluated its e ffects on cell cycle distribution when combined with DHA. The treatment of both cell lines with T-DM1 and with the DHA and T-DM1 combination inhibited cell-cycle progression, as indicated by the increased proportion of cells in G2/M phase (Figure 6a). Moreover, the treatment with T-DM1 induced a remarkable increase of cell population in sub-G1 area mainly in HCC1954 cells. The increase was even greater in HCC1954 cells exposed to the two-drug treatment, thus indicating apoptotic cells and/or fragmentation of DNA (Figure 6a).

An immunofluorescence analysis revealed aberrant mitotic spindles in both cell lines under all treatments. Both T-DM1 and the DHA and T-DM1 combination induced the formation of disorganized microtubule structures, unstructured tubulin foci, and multiple nuclei. In addition, a significant (approximately 2-fold) increase in density of microtubules was clearly found when HCC1569 cells were exposed to both DHA and DHA and T-DM1 combination, while we did not find any significant increase in density of microtubules in HCC1954 cells under all treatments (Supplementary Figure S4). Moreover, we also detected an aberrant distribution of phospho-TCTP in enlarged and morphologically altered HCC1569 and HCC1954 cells when treated with DHA and T-DM1 (Figure 6b).

Dividing cells with a defective mitotic apparatus undergo mitotic cell death/catastrophe [62]. Western blot analysis of cyclin B1 and phosphorylated histone H3 at Ser10 (p-Histone H3), two markers of M phase, showed that they were upregulated in HCC1569 cells, thus indicating that cells were arrested in mitosis. In addition, we found that activation of caspase 3 was induced by T-DM1 treatment. Notably, the increase of caspase-3 activity was even greater in cells exposed to DHA in combination with T-DM1, suggesting that cell death occurred in mitosis (Figure 6c). On the contrary, a di fferent pattern was found in HCC1954 cells. Indeed, no accumulation of cyclin B1 was found in both T-DM1 and DHA and T-DM1 treated cells. However, the percentage of sub-G1 phase were significantly increased. To confirm the induction of apoptosis, we evaluated the active caspase 3 and the proteolytic cleavage of PARP. Figure 6c shows that T-DM1 treatment induced a significant increase of cell death as evaluated by the amount of cleaved PARP, which was further increased upon T-DM1 and DHA treatment. As DNA damage triggers PARP activation [63], we evaluated the amount of DNA damage levels by studying the increase in the phosphorylation levels of histone H2AX. Intriguingly, a greater DNA damage was found in HCC1954 cells subjected to two-drug combination than those exposed to any single agent, as indicated by the increased levels of γH2AX (Figure 6c).

**Figure 6.** *Cont.*

**Figure 6.** The combination of DHA and T-DM1 induces mitotic catastrophe in HER2+ BC cell lines (**a**) Cells were pre-treated with DHA for 24 h and then treated with T-DM1 for 3 days. The bar graphs show the distribution of cycling cells. The histograms show the percentage of Sub-G1 cells of one representative experiment. Values represent the mean ± SD, *n* = 3. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. (**b**) Cells were treated as described in (**a**). The overlay of the three fluorochromes is shown (Merge): phospho-TCTP (red) and αTubulin (green) and nuclei stained with Hoechst (blue), bar = 25 μm (left side). A greater detail of the boxed area is shown, bar = 5 μm (right side). Data from representative experiments are shown. *n* = 2. (**c**) Western Blot analysis of the indicated proteins in cell lysates of cells treated as described in a). β-actin was used as loading control (left panel). For densitometric analysis, the intensity of each band was normalized to the respective β-actin (right panel). \* *p* < 0.05, \*\* *p* < 0.01.

Altogether, these data show that DHA in combination with T-DM1 induces severe mitotic defects and death in aggressive HER2+ BC cell lines.
