Tivozanib

Intuitive repositioning of an anti‑depressant drug in combination with tivozanib: precision medicine for breast cancer therapy

Naveen Kumar1 · Masoom Raza1 · Seema Sehrawat1

Abstract

Despite the existing therapies and lack of receptors such as HER-2, estrogen receptor and progesterone receptor, triple- negative breast cancer is one of the most aggressive subtypes of breast cancer. TNBCs are known for their highly aggressive metastatic behavior and typically migrate to brain and bone for secondary site propagation. Many diseases share similar molecular pathology exposing new avenues in molecular signaling for engendering innovative therapies. Generation of newer therapies and novel drugs are time consuming associated with very high resources. In order to provide personalized or preci- sion medicine, drug repositioning will contribute in a cost-effective manner. In our study, we have repurposed and used a neoteric combination of two drug molecules namely, fluvoxamine and tivozanib, to target triple-negative breast cancer growth and progression. Our combination regime significantly targets two diverse but significant pathways in TNBCs. Subsequent analysis on migratory, invasive, and angiogenic properties showed the significance of our repurposed drug combination. Molecular array data resulted in identifying the specific and key players participating in cancer progression when the drug combination was used. The innovative combination of fluvoxamine and tivozanib reiterates the use of drug repositioning for precision medicine and subsequent companion diagnostic development.
Keywords Drug repositioning · Combination therapy · Precision medicine · Companion diagnostic · Metastasis and TNBC
Abbreviations
HUVEC Human umbilical vein endothelial cells AKAP9 A-kinase-anchoring protein 9
Akt Protein kinase B ZO-1 Zonula occludens-1
TNBCs Triple-negative breast cancer cells

Naveen Kumar and Masoom Raza have contributed equally to this work.
Introduction
Cancer is known to be the second most common cause of public health worldwide. Out of the different types, breast cancer is known to be the foremost cause for the death in female population [1]. Cancer aggression accounts for the high mortality rate in patients. Triple-negative breast cancer (TNBC) is the utmost aggressive phenotype of breast cancer which shows high metastatic ability in visceral and central nervous system with poorer disease-specific survival [2, 3]. The absence of three specific receptors, namely estrogen (ER), progesterone (PR), and herceptin (HER-2) makes it

more resistant, and thus, the disease offers a very limited
opportunity for hormonal therapy [4]. Therapy limitations open new opportunities to study the molecular signaling in TNBC. Topical studies on TNBC at the molecular level have acknowledged new receptors that can be used as potential therapy targets [5]. Investigation of these molecular signal- ing led to involve epigenetic therapies, immunotherapies [6], and combined drug therapies (CDT) [7, 8].
TNBC metastasis is associated with lethal consequences even after the treatment, and in these consequences, tumor heterogeneity plays the profound role. Newer therapies

especially combination therapy are rapidly gaining popular- ity, and almost 80% of clinical trials are using combination therapy for TNBC treatment [9, 10]. Combination regime popularly targets different and important signaling pathways to reduce the cancer progression and have very low chances of cross resistance with each other [11]. These combination therapies works synergistically to inhibit cancer progression [12]. One of the interesting aspects of combination regime is that it helps in combating drug resistance as there is a less prospect for the tumor to develop resistance against numer- ous drugs simultaneously [12].
Although combination regime is widely utilized for targeting tumor progression, drug repositioning approach has been employed an addition in existing methods. This encompasses an already approved drug that can be tested for the treatment of other diseases by classifying their novel targets [13, 14]. Drug repositioning cuts the developmental risk and time, as repositioning candidates are industrialized only after numerous stages of clinical trials and are known to be much safer, along with a known pharmacokinetic profile. Precision medicine is used to characterize the customized medical treatment based on the patient’s own genetic profile, which includes new diagnostics and therapies. In particu- lar, advances such as cell sorting, epigenetics, proteomics, metabolomics, and more are converging with informatics and other technologies in a manner that is rapidly expanding the scope of this field. In recent studies, drug repositioning has been used for the treatment of glioblastoma [14, 15], ovarian cancer [16], cardio-renal diseases, and Alzheimer [17, 18].
In the present communication, we are reporting an inno-
vative combination of fluvoxamine, an anti-depressant molecule along with tivozanib for the treatment of TNBC. Tivozanib is a well-studied, potent, and selective vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI) and inhibits its phosphorylation even at pico- molar concentrations [19]. It inhibits other kinases as well, such as c-KIT and platelet-derived growth factor receptor (PDGFR). VEGFR is an ideal molecule for targeting tumor angiogenesis, and thus, the role of this drug has been widely exploited for the treatment of different types of cancers. It has been approved in European Union (EU), Iceland and Norway for the treatment of renal cell carcinoma and is currently in the phase III trial in order to get its approval worldwide [20]. It is under phase II trial for the treatment of glioblastoma [21], soft tissue sarcoma [22], and colorectal cancer [23]. On the other side of the study, our focus was on to exploit drug repurposing of fluvoxamine, an FDA- approved drug for the treatment of obsessive–compulsive disorder, anxiety disorder, and panic disorder. It belongs

to one class of antidepressants called selective serotonin re-uptake inhibitors (SSRIs) [24]. However, the molecular mechanism of action of Fluvoxamine on breast cancer is still unknown, we explored this possibility based on the available data on glioblastoma cytoskeleton effect [15].
Initially, this drug was administered to patients who were suffering from depression and anxiety related to cancer [25]. Further, some recent reports suggest that fluvoxamine acts as a potent inhibitor of actin polymerization which basi- cally affects the Akt/mechanistic target of rapamycin kinase (mTOR) and focal adhesion kinase (FAK) signaling and decreases the auto phosphorylation of FAK, thereby result- ing in disrupted actin reorganization and reduced migration. This suggests a possible role for Fluvoxamine in affecting the metastatic and invasive properties of cancer cells [26] and, thus, can be considered as a good candidate for repo- sitioning in TNBC. Recent advents in technology have also opened doors for advances in discovery biomarkers and their detection, to make prediction, diagnosis, and treatment evaluation more accurate, minimally invasive, and efficient. Herein, we are highlighting the synergistic effect of fluvoxamine and tivozanib in TNBC model system. We are depicting a significant reduction in the progression of TNBC cells after the treatment with combination regime. Early results on cytoskeleton distortion gave us the reasons to explore the oncologic suppression with this novel com- bination arrangement. We further worked on tumor migra- tion and invasive potential of cancer cells and found that the effect of combination was highly satisfactory. Tumor- induced angiogenesis is one of the crucial mechanisms for
the growth of tumor mass.
Angiogenic tubes formation was hampered in 2D in- vitro analysis. Later, we explored the key participants of metastatic behavior of TNBC cells, and the data showed the significant reduction in metastatic potential of TNBC. Flu- voxamine was an additional approach to analyze the tumor behavior, we also analyzed the stressed conditions induced by the fluvoxamine treatment. Treatment of Fluvoxamine along with tivozanib created a stressed environment in the tumor cells, and the stressed molecules were observed in proteome array analysis. Proteome array data give the new insights of participant in tumor inhibition.
Combination treatment of fluvoxamine and tivozanib to treat TNBC will be a new addition and has been previously unreported. Based on the mechanism of action mentioned in the literature for these drugs, we have hypothesized that combination of fluvoxamine and tivozanib will affect angio- genesis and metastasis in cancer cells. In this study, we have provided the proof of concept for drug repurposing along with its application in combination therapy for breast cancer.

Materials and methods
Reagents and antibodies

Trypsin, PBS (phosphate buffer saline), and skim milk were procured from HiMEDIA, India. Formaldehyde solu- tion, Triton-X-100 solution, and 2-mercaptoethanol were purchased from Sigma–Aldrich (USA). Cell culture insert (Cat No. MCEP12H48, Millipore), Calcein – AM (Cat No. 564061, BD Biosciences), and Matrigel (Cat No. 356237, BD Biosciences) were purchased. MDA-MB-231 cells were purchased from NCCS, India. L-15 Media (Cat No. Al011A) and FBS (RM10681) were acquired from HiMEDIA Labo- ratories India. Highly sensitive Supersignal™ West Pico Chemiluminescent Substrate (ThermoFisher, USA) was used for western blot. Fluvoxamine was obtained from Sigma-Aldrich (USA), and tivozanib was obtained from APExBIO. Antibodies purchased from commercial sources were MENA (Cat No. NBPI 87914, Novus), VEGFR (Cat No. PAB367Hu01, Cloud clone), Tubulin, AKAP9 (Cat No. NB100-86994, Novus), secondary anti-mouse (Cat No. 554002, BD, 1:2000), rabbit HRP (Cat No. 554021, BD,
1:2000), and Proteome Profiler Human Cell Stress Array Kit (Catalog # ARY018, R&D System, USA).
Cell culture methods

MDA-MB-231 cells were cultured in L-15 medium (HiME- DIA, India) supplemented with 10% FBS (Fetal Bovine Serum, Gibco, USA) and 1% Pen-Strep (1000 units/ml, Gibco, USA) and kept in 5% CO2 incubator at 37 °C. Trypsinization of MDA-MB-231 cells was done using Trypsin–EDTA (HiMEDIA, India). Cells were passaged twice a week; then cells were cultured in six and twelve well plates for experiments; and treatment was given at various concentrations of both the drugs. HUVECs were procured from Lonza and cultured in EBM-2 (endothelial cell growth basal medium) supplemented with 10% FBS, GA-1000, heparin, ascorbic acid, hEGF, R3-IGF-1, VEGF, hFGF, and hydrocortisone (as per manufacturer’s protocol).
Cell cytotoxicity assay

TNBC cell line were seeded into fresh 24-well plates with 15,000 cells/well. After cells were attached for 12–18 h, they were treated with fluvoxamine, tivozanib alone, and in combination with different concentrations for 24 h and 48 h. After 48 h of treatment, cells were harvested using 0.25% trypsin and counted with hemocytometer to get IC50 value of drug as compared to untreated control. After counting, we converted cell number into percentage, and then graph was made as percent reduction of cell number as compared

to the drug. The IC50 value of fluvoxamine is approximately 20 µM; for tivozanib, we got IC50 value around 6 µM; and for combination, we got less than 5 µM IC50 value. Rest of the experiment was done according to these concentrations only. In addition, we conducted more sensitive analysis of drug molecules on HEK 293T and MCF-7 cells. Cell viabil- ity was analyzed as discussed previously [15].
Wound healing assay

MDA-MB-231 cells were seeded in a 12-well plate (around 2.0 × 105 cells/well) with 2 mL complete media. After 24 h, treatment was done with subtoxic level of flu- voxamine (20 µM) [27] and subtoxic level of tivozanib (5 µM) [28] diluted in complete media separately and in combination. Assay was performed in triplicate. After treatment, scratch wounds were made by using 10 µl pipette tip. Cells were allowed to migrate into wounded area for 24 h, and then images were taken on Leica microscope. Images of 3–4 fields were taken per well to calculate the wound healing rate. The equation for the
calculation is %wound closure = [(initial wound width
−wound width 24 h after treatment with all drugs∕control)
∕initial wound width × 100.

Invasion assay

Cancer cells have the property to invade the surrounding tissue and metastasize to different parts of the body. We explored this property of cancer cells using transwell culture insert assay which is also called 2D invasion assay [29]. Invasion assay was performed using culture inserts (Corn- ing) for 12-well plate. Matrigel (BD) was coated onto the culture inserts overnight at 37 °C in CO2 incubator at con- centrations of 50 μg/mL/well. Drug-treated MDA-MB-231 cells were seeded in the top chamber with complete L-15 media. A total of 2.5 × 105 cells/ml in 200 µl media was seeded to each chamber. Lower chamber of insert was also filled with L-15 media (10% FBS). After 24 h, the cells were washed with 1X PBS. Fixing and staining were performed with methanol and crystal violet, respectively. Images of the invaded cells were captured by Leica Microscope from the lower chamber. A total of 3 random areas from each well were selected, and the experiment was repeated in triplicates.
Invadopodia detection and gelatin degradation assay

As our results indicates the reduction in invasive proper- ties of TNBCs, we explored further about the invadopodia detection after treatment with fluvoxamine and tivozanib.

Oregon green 488 was used to mount the coverslips homo- geneously. After the mounting with Oregon green, coverslips were washed thrice with prechilled glutaraldehyde (0.5% in PBS) and incubated with sodium borohydride (5 mg/ml in PBS) for 3 min at RT. Coverslips were washed with PBS containing penicillin (200 unit/ml). An equal number of fluvoxamine- and tivozanib-treated cells were cultured on the coated coverslips for 16 h. Later cells were fixed and permeabilized with 4% para-formaldehyde and 0.1% Triton- X-100, respectively. Cells were stained with phalloidin-actin for 30 min. All the coverslips were mount with anti-fade DAPI reagent, and images were taken and quantified and depicted in graphical representation.
Tissue culture media (TCM) collection

MD-MB-231 cells were seeded in 6-well plates and treated with fluvoxamine and tivozanib, alone and in combinations. Treatment was given for 48 h at 37 °C. Tissue culture media of control, fluvoxamine-treated, tivozanib-treated, and com- bination of fluvoxamine- and tivozanib-treated cells were collected and stored at − 80 °C. Later TCM was thawed and concentrated with the help of concentrator (3KDa Filter, Merck). The equal amount of TCM was used to incubate primary cells for further experiments.
Angiogenesis assay

HUVECs (around 5.0 × 103 cells/well) were seeded on matrigel-coated (10 mg/ml) 96-well plate with EBM media. Initially, matrigel was thawed on 4 °C and 100 μL was loaded in each well. 96-well plate were kept at 37 °C for 30 min. Primary cells were harvested and an equal number of cells were incubated on matrigel along with TCM of con- trol and treated MDA-MB-231 cells. Cell were seeded and incubated at 37 °C for 4 h. After 4 h, the images of tubes formed in each well, were carefully taken and analyzed with AngioTool. The tubes, branches and nodes were calculated and plotted.
Western blot analysis

TNBC cells were treated with fluvoxamine and tivozanib, alone and in combination for effective approach of cancer cell progression inhibition. Treated cells were analyzed for the expression of MENA, VEGFR, β-actin, and GAPDH. MDA-MB-231 cells were harvested and lysed with lysis buffer (0.5% SDS, 50 mM Tris–HCl, 1 mM EDTA). Protein separation was analyzed by 10 and 12.5% SDS-PAGE gel for 3 h at 120 V. Gel was transferred on to the PVDF membrane for 1 h, and membrane was blocked with blocking buffer (5% skim milk in PBS with 0.1% Tween-20) for 1 h. Pri- mary antibodies (material section) were incubated on to the

membrane in TBST O/N at 4 °C. Next day, respective sec- ondary antibodies were incubated. Membrane was imaged with Alpha Imager system (protein simple).
Immunofluorescence assay

MDA-MB-231 cells were seeded on a glass coverslip in a 12-well plate (around 2.0 × 105 cells/well) with 1 mL com- plete media. After 24 h, cells were treated with fluvoxamine (20 µM) and tivozanib (5 µM) diluted in complete media separately and in combination. After 24 h, cells were washed with ice cold 1X PBS twice, and then fixed with 4% para- formaldehyde solution for 10 min at room temperature. Cells were permeabilized with 0.1% Triton-X-100 in PBS for 10 min at room temperate. Blocking was done using FBS-PBS solution (1% FBS) for 2 h at room temperature. Immuno-staining of cells was done by incubating in pri- mary antibody overnight at 4 °C followed by incubation in their respective secondary antibody for 2 h. Coverslip was mounted using vector shield mounting reagent with DAPI and sealed. Slide was observed under fluorescence micro- scope (Eclipse Ti, Nikon).
Proteome array analysis

Stress proteome array analysis was conducted as described previously [43]. Briefly, TCM of combination regime-treated cells was collected and quantified. An equal amount of pro- tein from control cells and treated cells extract were loaded on each membrane for 24 h and developed as per manufac- turer protocol. The data were quantified with ImageJ.

Results
Combination regime of fluvoxamine and tivozanib targets breast cancer cell aggression via precisely targeting cytoskeleton and cellular adhesion

TNBC cells are well studied for high rate of metastasis in tumor microenvironment conditions. Cellular cytoskeleton members are the featured players for this step, and for this analysis, we used fluvoxamine and tivozanib. Initially, we determined the specific concentrations for fluvoxamine and tivozanib for the treatments. Cell counting analysis and CCK-8 analysis gave the specific concentration for the flu- voxamine, as there are no specific concentrations’ details available on breast cancer. We found the significant values on MCF-7 and HEK 293 T cells (Fig. S1). Fluvoxamine participates in many cellular activities, but its significant role in cancer cell progression or inhibition is still debat- able. Hayashi K et al. have shown the role of fluvoxamine in migration and invasion in glioblastoma [26]. We started

our initial analysis with cellular progression studies. Tivo- zanib is known for its VEGF-receptor inhibition activity and is also used for analyzing the cellular behavior. We found that tivozanib directly inhibits the cellular migration. Later, breast cancer cells showed a significant reduction after the combination treatment compared to control cells. A 24 h migration study gave the initial remark on the cancer cell migration (Fig. 1a).
To further confirm our data, we also looked on transwell migration studies. A number of migrated cells through transwell in combination treatment were very few in com- parison with control and alone treatment. Both the data complement each other and showed the significance of our study (Fig. 1b). To give the strength on our data, we also utilized another cell of breast cancer origin. We conducted the migratory analysis on MCF-7 cells and found significant reduction in cellular migration in alone and combination treatment (Fig. S2b) Cellular migration activities are associ- ated with cytoskeleton, cell adhesion properties, or cell cycle status. In addition, we also performed immunofluorescence studies to analyze the cytoskeletal and structural changes on cancer cells following treatment of fluvoxamine and tivoza- nib. Microtubules are part of the cellular cytoskeleton and participates in many cellular processes. We found that tubu- lin structure was deformed upon treatment with fluvoxamine and tivozanib. Combined effect of both the molecules was very different as compared to the control cells. Microtubule deregulation also plays a crucial role in cell migration prop- erties as previously reported. AKAP9 is known as a scaf- fold protein and a key player of cAMP signaling pathway. AKAP9 localization was observed at the nucleus and nuclear periphery in control cells but treatment of fluvoxamine and tivozanib modulates its distribution in cytoplasm (Fig. 1c, d).
Synergistic effect of fluvoxamine and tivozanib targets invasive potential of TNBCs

Cancer cells have the propensity to talk with the extracel- lular matrix (ECM) in order to metastasize for secondary propagation. In order to metastasize, cancer cells degrade the ECM and circumnavigate the surrounding environment. We drove on the invasive front of cancer cells to determine whether the present combination of drugs also affects the invasive capabilities of the MDA-MB-231 cells. We have observed that the presence of either of these drugs resulted in the reduced number of invading cells through the micro- pores of the culture insert as compared to control cells.
TNBCs are known for their aggressive behavior dur- ing metastasis, we found that a number of invading cells were comparatively very less after combined treatment of fluvoxamine and tivozanib. Combination regime effect on invasion reduction is may be due to the inactivation of

proteases in the cells as they are known for digesting the surrounding tissue and help in migration. The combination of drugs demonstrates approximately fourfold decrease in invasion compared to control (Fig. 2a). We also quantified the invaded cells with optimal density method, and results were similar (Fig. 2b). To give further strength to our results, we also investigate the invasive potential of MCF-7 cells. Alone and combination treatments were very effective and combination regime gave a significant reduction of invasive cells (Fig. S2a).
Combination regime drastically targets tumor microenvironment

To further explore the metastatic potential of TNBCs, we performed invadopodia detection assay. Actin protrusions form while traveling to the distant part during metastasis. We found that Oregon Gelatin 488 was degraded in control cells, which is a symbol of generalized cellular metastasis. We found gradual reduction in actin protrusions after treat- ment of tivozanib and fluvoxamine. Combination showed a smaller number of degraded protrusions which suggest that TNBCs metastatic potential is being compromised (Fig. 3a). Degradation was analyzed and quantified. This shows that the combination of fluvoxamine and tivozanib reduces the EC degradation in breast cancer cells (Fig S3). Tumor mass highly regulated by surrounding environment conditions and these microenvironment conditions promote induction of nearby capillaries to generate small vessels to satisfy the need of tumor cells.
Tumor cells can feed through these small capillaries and use them for metastasis. The ability of primary endothelial cells to form angiogenic tubes in the presence of the TCM of treated cancerous cells was determined through tube forma- tion assay on primary HUVECs. Small branched tubes were generated while using TCM of control cells but treated cells media showed a significant reduction in nodes and tubes. In addition, combination drug-treated TCM depicts a huge inhibition in tube formation. These data suggest that TNBCs failed to release specific growth factors to induce primary cells to make tube-like structure. Combination approach of fluvoxamine and tivozanib showed a significant reduction in angiogenic tube formation (Fig. 3b).
Combination treatment effectively regulates expression of metastatic markers: mechanistic insight into precision medicine‑based approach

Combination regime of fluvoxamine and tivozanib effec- tively targets key players of metastasis. We have defined the characteristics of TNBC after the treatment of combina- tion in our previous sections. MENA is known as a metas- tasis marker which regulates cellular movement and also a
Fig. 1 Synergistic effect of fluvoxamine and tivozanib targets cellu- lar architecture and inhibits motility: a TNBCs exhibit the property of migration from the primary sites. We explored this interesting phe- nomenon by wound healing analysis. After a run of 24 h, treated cells showed significant reduction. b Transwell migration data supported the wound healing data and showed effect of combination treatment. c Microtubules are one of the most crucial parts of cytoskeleton, which help in cell division and movement. We found that microtu- bules were disorganized while treatment with our drug molecules (a’-d’), which can be an important reason for migration inhibition. AKAP9 is a scaffold protein and a key player in cAMP signaling and cancer progression. We found the localization of AKAP9 distorted in treated cells. (a’- d’) enlarged view of all the images

participant of actin polymerization assembly. Cellular motil- ity is determined by actin stability or alternatively regulated by MENA. Expression of MENA was reduced in combi- nation treatment (Fig. 4a). Cellular imaging data showed the gradual decrease from alone to combination treatment. We quantified the data and found the significant changes in intensity profile of MENA and occludin (Fig. S4). We further confirmed this with western blot analysis (Fig. 4b). To explore the migratory potential factors, we explored occludin level in treated cells. Tight junctions are the cru- cial factors in cellular motility as occludin plays vital role in cellular polarity and paracellular assembly. Expression of occludin is found to be different in different cancers [30–33]. Although the expression of occludin is stable in treated cells compare to control cells, however, the effect on motility was less significant. There could be some additional factors along with occludin which needs to be targeted and explored further for metastatic inhibition. Interestingly, we also observed the downregulation of VEGF-R2. VEGF-R2 participates in many molecular mechanisms of cancer pro-
gression (Fig. 4 b).
To further explore the cellular motility, we observed the impressions of GM130. GM130 is Golgi matrix protein and regulates cellular polarity and directions [34, 35]. Control cells showed systematic alignment of GM130 in cytoplasm which shows its effect on tumor cells directional motility. Combination of fluvoxamine and tivozanib disturbs the GM130 level in cytoplasm and affects the cellular direc- tional movement, which helps in metastasis (Fig. 4c). Later, our observations showed the significant reduction in actin assembly. As MENA regulates the actin assembly [15], there could be a possible effect of MENA inhibition on actin polymerization. This leads to target the metastatic potential of TNBC cells (Fig. 4c). All the images were quantified and analyzed with NIS and Image J. (Fig. S4).
Stress markers activated upon synergistic effect of fluvoxamine and tivozanib

It is expected that stress is the result of inhibition of cellular migration and invasion of tumor cells. We wanted to analyze

the stress response subsequent to the treatment of our com- bination regime. Proteome array analysis showed that the key participants of stress response were highly affected with combination treatment. Very specific molecules were observed under stress conditions. One of the major mol- ecules is HIF-1 alpha, which is responsible for malignancy in tumor cells [36, 37].
Combination regime targeted its expression which shows the inhibition of metastasis. Stress conditions alter the P53 level via post-translational modifications but its role is still not understood in terms of tumor response [38]. However, phosphorylated P53 does not change during the treatment. Heat shock protein – HSP-70 expression, is generally enhanced in breast cancer tissue [39]. HSP-70 expression is associated with malignancies in breast cancer, and we found a significant reduction in combination treatment (Fig. 5a, b). We analyzed the complete array and select few specific tar- gets and showed as outstanding results. Rest data of molecu- lar proteins are analyzed and represented in Fig S5. Quantifi- cation was done by ImageJ. The proteome array data showed that a number of important and interesting molecules were modulated upon the combination treatment with fluvoxam- ine and tivozanib. However, their specific implication with the respective drug molecules needs to be further explored. Schematic representation depicts the proposed mechanism of action of fluvoxamine and tivozanib (Fig. 5c).

Discussion
Our study has provided a new insight and proof of concept to understand the role of combination therapy along with drug repositioning strategy in combatting TNBC. In this study, we have demonstrated that these two strategies are very effective in targeting migration, angiogenesis, and metastatic axes of TNBC cells. It is known that enhanced migratory potential of cancer cells is an important therapeutic target for TNBC. The scratch wound migration assay performed using MDA- MB-231 cells indicated an impaired migration of cells after treatment with fluvoxamine and tivozanib individually. How- ever, the inhibition observed in combination therapy was far more significant. Moreover, the percentage wound healing was negative in case of combination therapy, indicating a dual effect of reduced migration and cell survival on treat- ment (Fig. 1a).
Immunofluorescence imaging showed aberrant organiza- tion and overall distortion of tubulin microtubules in can- cer cells after treatment with combination of fluvoxamine and tivozanib (Fig. 1c). As a cytoskeletal element, tubulin is also involved in cancer metastasis and migration. Acety- lated α-tubulin is found to be highly expressed in metastatic breast cancers, and it has been demonstrated that βIII-tubulin

 
Fig. 2 Combination of fluvoxamine and tivozanib inhibits the inva- sive properties of TNBCs: a TNBCs invade the surrounding tissue to metastasize. Here, we mimic the invasion conditions in a 2D for- mat. Culture insert was used for this mimicking. Interestingly, flu- voxamine showed less number of invaded cells along with tivozanib, combination treatment reduced the numbers of invaded cells, and it showed a drastic change in invaded cell number. b To further con-firm the impact of combination, we imaged the membrane of culture insert and take the absorbance of attached cell in the membrane. We found significant effect of combination of our drug molecules similar to culture insert data. These data support our previous data of cellular migration inhibition. Data were quantified and showed in graphical representation. Scale bar is 100 microns

plays an important role in the regulation of breast cancer- associated brain metastasis [40, 41].
AKAP9 is an important scaffolding molecule involved in the regulation of microtubule dynamics and specifically the epithelial-mesenchymal transition, which precedes invasion in cancer cells [42–44]. Both in-vitro and in-vivo conditions, AKAP9 has been demonstrated to be associated with cancer metastasis, invasion, and proliferation. AKAP9 is also typi- cally up-regulated in cancer tissues as compared to normal cells [45]. Through fluorescence microscopic analysis, we found that the typically AKAP9 is delocalized upon treat- ment by subtoxic level of fluvoxamine and tivozanib war- ranting further exploration in this protein (Fig. 1c). These observations indicated that combination therapy of these
drugs has an appreciable effect on the migration potential of MDA-MB-231 cells. In addition, similar results were observed when the effect of fluvoxamine and tivozanib treat- ment was elucidated for invasive properties of TNBC cells. There was a significant reduction in the number of MDA- MB-231 cells that were able to invade through in-vitro micro-pores after treatment with combination of fluvoxam- ine and tivozanib (Fig. 2a). Vascular invasion in primary tumor along with migration not only is an indication of aggressive cancer subtypes but also implies poor progno- sis for breast cancer [46]. These results point towards an overall decrease in the metastatic properties of TNBC post-
treatment with fluvoxamine and tivozanib.

Fig. 3 TNBCs-induced angiogenic potential inhibited after treatment with fluvoxamine and tivozanib: a combination of fluvoxamine and tivozanib affects the protrusion formation in TNBCs, which supports the idea of metastasis inhibition. The high number of actin protru- sions showed high rate of metastasis. b TNBCs secrete growth factors in tumor microenvironment to activate angiogenic potential of pri-
mary cells and to satisfy their functional needs. We found that tivo- zanib and fluvoxamine target and inhibit the secretion of growth fac- tors required for angiogenesis comparative to non-treated cells. Graph showed the number of tubes formation and number of nodes forma- tion in 2 D angiogenesis assay, respectively. Arrow head represents the number of nodes and star represent the number of tubesSprouting and the subsequent angiogenesis, coupled with migration and invasion, are some of the intermediate steps in cancer metastasis. Since intra-tumor microvascular density (iMVD) is an important factor that is involved in survival of cancer cells [47], we hypothesized a significant effect on cancer angiogenesis under the effect of drugs. In the present study, tube formation assay was performed in order to assess the effect of combination therapy on the angiogenic potential of primary endothelial cells. Tube formation was observed to decrease significantly upon treatment by the combination of drugs (Fig. 3b).

A significant decline in the number of tubes and nodes in HUVECs were observed after 24 h treatment with combina- tion therapy drugs as compared to both control and single drug treatments. In previous studies on tumor, stimulation with angiogenic inhibitory molecules results in an overall decrease in metastasis and growth [48]. Clinical studies have also elucidated the role of angiogenesis in breast can- cer migration and metastasis, indicating that high micro- vascular density (MVD) is associated with highly invasive carcinomas and a significant up-regulation in VEGF signal- ing [49, 50]. Our results, therefore, confirm the efficacy of

targeting tube formation and demonstrate the merit of the present combination therapy in targeting cancer-associated angiogenesis.
MENA participates in cancer cell migration and is known as a metastatic marker [47, 51]. Studies showed that high expression of MENA is directly correlated with poor out- comes in breast cancer patients. Our data showed a sig- nificant reduction in MENA expression upon treated with fluvoxamine and tivozanib, and this effect was enhanced when both the drugs affect synergistically. We also found the inhibition of VEGFR, which is responsible for tumor- induced angiogenesis (Fig. 4b). MENA is also known for regulating acting assembly and polymerization. We found a change in actin as well. This shows the specific targeting of cytoskeleton molecules which can be a crucial way of target- ing migratory potential of tumor cells (Fig. 4c).
We observed in our stress protein array analysis that the combination therapy approach targets many interesting sign- aling molecules. The mechanism of action of fluvoxamine and tivozanib has been clearly demarcated using the pro- teome array analysis. The conclusive model of combina- tion therapy involving both these drugs is highlighted and

Fig. 4 Fluvoxamine and tivozanib target the metastatic markers in combination treat- ment: a immunofluorescence studies showed the changes in expression of MENA and occludin compared to control cells. MENA and occludin play a crucial role in tumor
metastasis. b Western blot data showed the inhibition of MENA which is known as a potential marker of metastasis. We also found the reduced expression
of VEGF-R2 which helps in tumor-induced angiogenesis. Our data suggested that com- bination of fluvoxamine and tivozanib reduced the metastasis and tumor-induced angiogen- esis. c Later, we also found a prominent effect on actin and GM130. Both the molecules are key factor in tumor motility shows the mechanism of probable action taking into account all our experimental conclusions (Fig. 5c). Combination therapy along with repurposing drugs opens new venues
for developing novel strategies for the precision therapy. These molecules can be further targeted precisely along with

Fig. 5 Stress analysis explains the key proteins involved in tumor pro- gression: a array analysis provided the details of stress response dur- ing our treatment of combination. b Data quantified and represented.

existing therapies for precision therapy and companion diag- nostic development.
Statistical analysis

Angiogenesis data were analyzed by one-way ANOVA test using GraphPad Prism Software, La Jolla California USA. Migration, invasion, and transwell migration assay were ana- lyzed by unpaired t test using GraphPad Prism Software, La Jolla California USA. Results were considered as statisti- cally significant when P < 0.05. Proteomic array and west- ern blots were analyzed by ImageJ. Confocal microscopy images’ quantification (mean intensity) is done by software NIS 4.3.
Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s11010-021-04230-1.

Acknowledgements Seema Sehrawat acknowledges the research asso- ciate fellowship of Dr. Naveen Kumar by her DST-SERB grant. Shiv Nadar Foundation is acknowledged for PhD fellowship to Mr. Masoom Raza.

c Schematic representation of effect of combination of fluvoxamine and tivozanib in TNBC cells
Funding This work is supported with Department of Science & Tech- nology, Science and Engineering Research Board (DST-SERB) Grant (EMR/2017/003312), Government of India.

Declarations

Conflict of interest The authors declare no conflict of interest.

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