Lurasidone Sensitizes Cancer Cells to Osimertinib by Inducing Autophagy and Reduction of Survivin

Background/Aim: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are key drugs in cancer treatment due to their minor adverse effects and outstanding anticancer effects. However, drugs for overcoming EGFR-TKI resistance are not in clinical use so far. Therefore, to overcome resistance, we focused on lurasidone, a new antipsychotic drug, due to its mild adverse effect profile from the viewpoint of drug repositioning. Materials and Methods: We explored the effects of lurasidone alone or in combination with EGFR-TKI on the growth of osimertinib-resistant cancer cells the anti-apoptotic marker expression such as survivin, and autophagy levels by LC-3B expression. Results: Within a non-toxic concentration range in normal cells, lurasidone and osimertinib combination therapy showed a growth-inhibitory effect in osimertinib-resistant cancer cells in vitro and in vivo. Furthermore, lurasidone decreased survivin expression and mildly induced autophagy. Conclusion: Lurasidone may increase the sensitivity to osimertinib in osimertinib-resistant cancer cells in drug repurposing.

Immunoblot analysis. Cells were washed with PBS and lysed in RIPA buffer [10 mM Tris-HCl (pH 7.4), 0.1% SDS, 0.1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1.5 mM Na3VO4, 10 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, and 1% protease inhibitor cocktail set III (Sigma)]. After centrifugation for 10 min at 14,000 × g at 4˚C, the supernatants were harvested as the cell lysates, and the protein concentration of the cell lysates was measured using a BCA protein assay kit (Thermo Fisher Scientific). Cell lysates containing equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were probed with primary antibodies followed by an appropriate HRP-conjugated secondary antibody according to the manufacturer's protocol. The immune-reactive bands were visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
Immunofluorescence analysis. The protocol of the immunofluorescence analysis was modified from a previous study (18). In brief, A549 cells were seeded on coverslips in 35-mm dishes and used in the experiments. After the cells were fixed with 4% (w/v) paraformaldehyde at room temperature (RT) for 10 min and washed with PBS three times, they were permeabilized and blocked with 0.4% Triton X-100/2% FBS in PBS at RT for 10 min. After being washed with PBS three times, the cells were incubated with a primary antibody in PBS containing 2% FBS at RT for 60 min and then incubated with Alexa Fluor 488-conjugated secondary antibody (A11034, Thermo Fisher Scientific) and Hoechst 33342 (10 μg/ml) in the same buffer at RT for 10 min. Fluorescent images were acquired using a confocal laser-scanning microscope (FLUOVIEW FV10i: OLYMPUS, Tokyo, Japan).
Mouse study. The mouse xenograft study was carried out as previously described (14,19). After anesthetization (intraperitoneal injection of medetomidine, midazolam, and butorphanol at 0.3 mg, 4 mg, and 5 mg per kg, respectively), A549 CSLCs (1×10 5 ) suspended in 200 μl PBS were implanted subcutaneously in the flank region of 7-week-old male BALB/cAJcl-nu/nu mice (CLEA Japan, Inc., Tokyo, Japan). The tumor volume was assessed by measuring the tumor diameters using calipers and calculated as the larger diameter × smaller diameter × smaller diameter. For the systemic administration of drugs, stock solutions of lurasidone (2 mg/ml) and osimertinib (2 mg/ml) were diluted in DMSO to prepare 150 μl solutions for each injection. Lurasidone was administered by oral gavage to mice at 10 mg/kg five times a week and osimertinib was orally administered at 5 mg/kg also five times a week. The drug treatment was started 12 days after tumor implantation and confirmation of subcutaneous tumor formation (approximate average: 25 mm 3 ), and similar-sized-tumor-bearing mice were randomized into four groups before the initiation of drug treatment. All animal experiment protocols were approved by the Animal Research Committee of Yamagata University.
Statistical analysis. The results are expressed as the means and standard deviation (SD). The differences were compared using the two-tailed t-test. p-Values <0.05 were considered significant and indicated with asterisks.

Results
Lurasidone sensitizes various cancer cells to osimertinib. We first examined whether lurasidone sensitizes cancer cells to osimertinib. Various cancer cells, cancer stem cells, and patient-derived glioma cells were treated with lurasidone and osimertinib to measure cell viability. As shown in Figure 1, osimertinib-resistant cancer cells, cancer stem cells, and patient-derived glioma stem cells were sensitized by the addition of lurasidone. We then treated normal human fibroblast IMR-90 cells with lurasidone and measured their viability in order to examine the cytotoxicity of lurasidone against normal cells. As shown in Figure 2, lurasidone had little toxicity against normal cells. These findings indicate that lurasidone sensitizes various cancer cells, cancer stem cells, and patient-derived glioma cells to osimertinib in vitro without negatively impacting normal cells.
Lurasidone sensitizes cancer cells to osimertinib by reducing the expression of survivin. Next, we examined the mechanisms by which lurasidone sensitizes cancer cells to osimertinib. A number of molecular and/or cellular mechanisms are involved in determining the sensitivity and resistance to EGFR-TKIs; these mechanisms include increased expression of c-MET (20), emergence of resistance genes (21), modulation of autophagy (18) and expression of survivin, an anti-apoptotic molecule (22,23). We previously demonstrated that cells can be sensitized to EGFR-TKIs via reduced expression of survivin (13,14). Thus, we focused our examination on the expression of survivin. Specifically, various cancer cells, cancer stem cells, and patientderived glioma cells were treated with lurasidone alone, and the expression of survivin was examined by immunoblotting. As shown in Figure 3, the expression of survivin was reduced when cells were treated with lurasidone. We also knocked-down the expression of survivin in A549 lung cancer cells and subsequently treated them with osimertinib to measure cell viability. As shown in Figure 4, the expression of survivin was reduced and sensitivity to osimertinib was elevated. Lastly, cells were treated with osimertinib and YM155, a pharmacological suppressor of survivin, to measure cell viability. As shown in Figure 5, the expression of survivin was reduced and sensitivity to osimertinib was elevated.

Lurasidone partially sensitizes cells to osimertinib by increasing autophagy.
Since we previously demonstrated that increased autophagy led to the sensitization of cells to EGFR-TKIs (18), we treated various cells with lurasidone alone and performed immunoblotting to examine the level of autophagy. As shown in Figure 6A, treatment with lurasidone led to an increase in LC3B expression. Similarly, immunostaining of cells after lurasidone treatment revealed a localized increase in cytoplasmic staining ( Figure 6B). Cells were also treated with an autophagy inhibitor, 3-MA, to partially block autophagy induced by lurasidone, and were subsequently treated by osimertinib to measure cell viability. As shown in Figure 6C-D, there was a slight reduction in the sensitivity of cells to osimertinib. Collectively, these findings indicate that lurasidone may sensitize cells to osimertinib by increasing autophagy.
Lurasidone sensitizes tumors to osimertinib in vivo. Our in vitro results indicated that lurasidone enhances the sensitivity of cells to osimertinib by mechanisms such as the reduction of survivin expression. Thus, an animal study was performed in order to determine whether the findings in vitro can be replicated in vivo. A549 CSLCs were injected subcutaneously in both sides of the flanks of nude mice. After tumor formation, the mice were categorized into 4 groups ensuring that the average tumor size was similar across the groups. After administration of lurasidone and osimertinib, we monitored the size of the tumors as well as the overall health of mice including body weight. As shown in Figure 7, administration of the combination of lurasidone and osimertinib was effective in suppressing tumor growth compared to individual drugs alone. Although a nonsignificant weight loss was observed soon after osimertinib treatment, the weight recovered relatively early during the observation period. These findings indicate that lurasidone sensitizes cancer cells to osimertinib in vitro as well as in vivo.

Discussion
EGFR-TKIs are considered one of the most important antitumor agents as they are highly effective and only cause mild adverse events. In particular, a third-generation EGFR--TKI, osimertinib, is used as a worldwide standard due to its efficacy and cytotoxicity (4,5). However, osimertinib is not effective in certain cancer types, such as A549 lung cancer with wild-type EGFR, and other histological types that are resistant to osimertinib. Thus, there is a need for a novel treatment strategy to overcome resistance to osimertinib in EGFR-TKI-resistant cancer types. Lurasidone is a clinically approved drug and widely used against bipolar disorder and schizophrenia as it is well-tolerated. It was particularly designed to reduce side-effects as it acts as a 5-HT 7 antagonist and has a low affinity to H1 (8,24). Thus, it may also be well-tolerated in frail cancer patients. Furthermore, since cancer patients often suffer from depressive symptoms (9), lurasidone may be proposed as a treatment strategy aimed at improving the physical and mental health of cancer patients as it can have both anticancer and anti-depression effects. Notably, it is already approved for clinical use and is an attractive target for drug repositioning (repurposing), which is an inexpensive strategy requiring a relatively short time until approval is granted for another indication in clinical practice (25,26). Collectively, lurasidone may be an attractive option for the treatment of cancer. In the present study, we used an animal model to evaluate the antitumor effect of lurasidone in combination with osimertinib. When we take the human equivalent dose [HED=animal dose in mg/kg • (animal weight in kg/human weight in kg) 0.33 ] into account (27), the dose of lurasidone (10 mg/kg) administered to the mice (~25 g body weight) in this study corresponds tõ 0.77 mg/kg in humans (~60 kg body weight), which is within the range of the clinically used dose for humans (20-60 mg/day: 0.33-1 mg/kg for humans with 60 kg body weight). Thus, in this study, lurasidone exerted an antitumor

. Lurasidone sensitizes various tumor cells to osimertinib. The indicated cancer cells were cultured with or without 2 μM osimertinib (OSI) and with or without 5 μM lurasidone (Lura) for 3 days (lurasidone concentration was 3 μM for GS-NCC01), and then subjected to cell viability assay using trypan blue. The initial cell number was 1×10 5 cells. The total number of cells (viable and dead) (left panels) and percentage of dead cells (right panels) are shown. Values represent means±SD from quadruplicate samples of a representative experiment repeated three times with similar results. *p<0.05. In the left panels, the number of viable cells was compared.
effect in mice at a clinically relevant dose in combination with osimertinib.
To our knowledge, there are no studies to date that demonstrated the possibility of repurposing lurasidone for the treatment of cancer. While our findings were novel, we were unable to determine the detailed molecular mechanisms of action. Although our analysis may not be complete, our findings suggest that one of the possible mechanisms by which lurasidone sensitizes cancer cells to osimertinib is via reduction of survivin expression.
Resistance to EGFR-TKIs, such as osimertinib, is mediated by various mechanisms including increased expression of c-MET (20). The role of survivin, one of the anti-apoptotic factors, has been also indicated to mediate resistance (22,23). Survivin is increasingly being recognized as an important target for cancer treatment, and a number of preclinical and clinical studies are currently being conducted. Recently, the combination of first-generation EGFR-TKI (erlotinib) and a small molecule inhibitor of survivin (YM155) was tested in a clinical trial and its initial results are promising (28).
Our findings also suggested that lurasidone partially sensitizes cancer cells to osimertinib by increasing autophagy. Autophagy can have various roles as it may be suppressed or increased depending on the phase of tumor formation and the actual state of the tumors (29,30). In accordance with our findings, some studies suggest that increased autophagy leads to cell death following EGFR-TKI treatment (18,31). However, other studies suggest that inhibition of autophagy leads to sensitization of cells to EGFR-TKIs (32)(33)(34), and that EGFR-TKIs activate autophagy to suppress cell death (35). Thus, there is no consensus as to how EGFR-TKIs and autophagy interact. Our findings demonstrated limited involvement of autophagy; thus, future studies are needed to better understand this possible mechanism.
Lurasidone targets a wide spectrum of receptors for biogenic amines, including serotonin, dopamine, and      adrenaline. Therefore, it is difficult to determine the specific molecular mechanisms of lurasidone's anticancer effects. We have reported that brexpiprazole, an antagonist of D2 dopaminergic receptor, suppresses the expression of survivin and sensitizes cancer cells to osimertinib (13). Meanwhile, we have reported that doxazosin, an alpha 1adrenergic receptor antagonist, induces autophagy and sensitizes cancer cells to osimertinib (18). Because these drugs in our past reports share the receptor targets with lurasidone, survivin suppression and autophagy induction by lurasidone might be mediated by the inhibition of D 2 and alpha 1-adrenergic receptors, respectively although further studies are required.
To our knowledge, there has been little evidence in the literature to suggest an association between autophagy and survivin, which is an anti-apoptotic molecule. Nevertheless, some studies demonstrated that reduced expression of survivin leads to activation of autophagy in some cancer types (36)(37)(38). Other studies demonstrated that autophagy and survivin independently modulate therapeutic effects (39), and that autophagy suppresses the expression of survivin (40). Although it seems unlikely that there is a direct association between survivin and autophagy, future studies are needed as they are both promising targets for cancer treatment.
There are several limitations to this study. Importantly, we were unable to identify particular receptor signaling pathways among the many points of action of lurasidone that play an important role in sensitizing cells to osimertinib. This was due to the difficulty in obtaining agonists and antagonists that are specific to receptor subtypes, such as 5-HT 7 agonists and antagonists, as they have an unknown mechanism of action in humans. Furthermore, it is difficult to reproduce the reaction and block the expression in neurotransmitters such as dopamine and 5-HT. The points of action of lurasidone may be off-target and not restricted to neurotransmitter receptors. Thus, further examinations are required.
In conclusion, our findings in vitro and in vivo suggest that lurasidone may sensitize various cancer cells to osimertinib. Reduced expression of survivin and increased autophagy may play a role in sensitizing cells to osimertinib.

Conflicts of Interest
The Authors declare no conflicts of interest.