AAA ATPase TRIP13 is overexpressed in prostate cancer and promotes tumor progression

Highlights

• •Integrative analysis of multiple data showed that TRIP13 is overexpressed in PCa.
• •Small percentage of PCa showed amplification of the TRIP13 locus.
• •TRIP13 is critical for PCa proliferation and invasion.
• •Inhibition of TRIP13 by DCZ0415 reduced PCa proliferation and tumor growth.
• •TRIP13 represents a potential therapeutic target in TRIP13-overexpressing PCa.

Abstract

Prostate cancer (PCa), a common malignancy, is a leading cause of cancer-related deaths among men. Advances in high-throughput technologies have led to the identification of various genetic alterations, including amplifications, deletions, mutations, gene fusions, and aberrant gene expressions, associated with PCa initiation and progression. Identifying key drivers of tumor progression and their underlying signaling pathways contributes to early diagnosis and therapeutic targeting. Here, we showed that thyroid hormone receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family is overexpressed in PCa. Additionally, we observed amplification of the TRIP13 locus in a small subset of PCa samples. Functional studies demonstrated that TRIP13 knockdown in PCa cells reduced their proliferation and invasion. Furthermore, ectopic overexpression of TRIP13 in prostate epithelial cells (RWPE-1) resulted in enhanced cell invasion. Additionally, pharmacologic inhibition of TRIP13 by the small molecule inhibitor DCZ0415 suppressed PCa cell proliferation, induced apoptosis, modulated markers of the epithelial-mesenchymal transition (EMT), and inhibited tumor growth. Overall, these findings highlight a functional role for TRIP13 in PCa progression and demonstrate its potential as a therapeutic target in TRIP13-overexpressing PCa.

Introduction

Prostate cancer (PCa), a leading cause of cancer-related deaths in men, shows genomic heterogeneity and various molecular alterations. These diverse molecular changes suggest the involvement of multiple signaling pathways during PCa progression [1]. In early, localized stages, PCa is typically androgen receptor (AR)-dependent and is effectively managed by anti-androgen therapies. More aggressive, metastatic castration-resistant PCa patients are treated with second-generation drugs, including AR antagonists such as enzalutamide and androgen biosynthesis inhibitors like abiraterone [2,3]. However, over time, drug resistance develops, and some tumors progress to a metastatic stage, at which they are essentially incurable. Therefore, to improve patient outcomes, an understanding of the mechanisms underlying PCa initiation, progression, and metastasis is necessary for identifying new therapeutic targets.

Advances in genomic technologies have allowed the generation of large-scale sequencing datasets and led to the identification of cancer-specific molecular signatures and pan-cancer alterations. In the present investigation, by analyzing publicly available microarray and the next-generation transcriptome sequencing data, we identified overexpression of thyroid hormone receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family [4].

TRIP13 interacts with hormone-dependent transcription factors, suggesting a role in PCa cancer biology and showing its potential as a therapeutic target [5]. TRIP13 are implicated in mitotic regulation, DNA damage repair, and therapy resistance [[6], [7], [8]]. Proteomic analyses show that TRIP13 interacts with p31^comet, a spindle assembly checkpoint silencing protein, and that this interaction is conserved in mice and humans [[9], [10], [11]]. TRIP13 also interacts with other mitotic regulators and is involved in organizing the meiotic chromosome axis, regulating repair of double-strand breaks [12], and facilitating meiotic recombination by influencing crossover and non-crossover pathways. It is essential for the removal, from synapsed chromosomes, of HORMA domain-containing protein 1 (HORMAD1) and HORMAD2 [4].

TRIP13 also interacts with Charcot-Marie-Tooth disease type 2 (CMT2)-associated proteins [11], which are involved in the spindle checkpoint, a key contributor to chromosome integrity [13]. Dysregulation of this checkpoint can lead to aneuploidy, a hallmark of cancers. We have previously shown that TRIP13 is overexpressed in head and neck [14], colorectal [15], and pancreatic [16] cancers, in which it functions in DNA repair and tumor progression. Moreover, TRIP13 is targetable with the small molecule inhibitor DCZ0415, which suppresses tumor growth and metastasis [17].

In the current study, we report that TRIP13 was overexpressed both in primary and metastatic PCa, as demonstrated by integrative transcriptomic analyses, immunohistochemical staining (IHC), and immunoblotting. We further delineated the mechanistic role of TRIP13 in PCa, showing that its overexpression enhanced invasion of prostate epithelial cells (RWPE-1). Pharmacological inhibition of TRIP13 with DCZ0415 reduced cell proliferation, induced apoptosis, modulated markers of the epithelial-mesenchymal transition (EMT), and suppressed tumor growth. These findings suggest that TRIP13 contributes to PCa progression and may serve as therapeutic target for TRIP13-overexpressing prostate tumors.

Materials and methods

Cell lines

PCa cell lines (DU145 and PC3) purchased from the American Tissue Culture Collection (Manassas, VA, USA) were cultured in media (EMEM for DU145 cells and RPMI 1640 for PC3 cells) supplemented with 1% penicillin-streptomycin and 10 % fetal bovine serum. RWPE-1 cells obtained from ATCC were cultured in K-SFM containing bovine pituitary extract (BPE, 50 µg/ml) and epidermal growth factor (EGF, 5 ng/ml). These cells were kept at 37 °C with 5% CO₂ in a cell culture incubator.

PCa tissues

For this study, we utilized patients’ tissues with localized PCa who have undergone radical prostatectomy at the University of Michigan Hospital. Tissue samples were also from androgen-independent metastatic PCa patients involved in a rapid autopsy program as described earlier [18,19]. The clinical and pathological data were maintained in a secure database. From all human subjects informed consent was obtained through the University of Michigan Medical School Institutional Review Board. This study was approved by the Institutional Review Board at the University of Michigan Medical School

Expression analysis

TRIP13 gene expression data were acquired from the cancer genome atlas (TCGA) transcriptome sequencing and analyzed using UALCAN cancer data analysis platform (https://ualcan.path.uab.edu) [20].

For immunoblot analysis, protein lysates (20 μg) from normal and PCa cancer tissues, as well as PCa cells, were separated by using NuPAGE 4–12% Bis–Tris Midi Protein Gels, (Thermo Fisher Scientific, Waltham, MA, USA), and transferred onto polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). These membranes were then incubated with primary antibodies overnight for TRIP13 (19602-1-AP, Proteintech, IL, USA), proliferating cell nuclear antigen (PCNA, 10205-2-AP, Proteintech), cleaved caspase 3 (9664, Cell Signaling Technology, MA, USA), N-cadherin (13116, Cell Signaling Technology), E-cadherin (3195, Cell Signaling Technology), and vimentin (5741, Cell Signaling Technology), followed by incubation with secondary antibody conjugated with HRP (SA00001-2, Proteintech) at room temperature for 1 hr. HRP-conjugated β-actin monoclonal antibody (HRP-60008, Proteintech) was used as a loading control. The signals were detected by Immobilon Classico western HRP substrate (Millipore, MA, USA) using Amerson Imager 600 (GE Healthcare, NJ, USA). The relative intensity of immunoblot bands was quantified be ImageJ (https://imagej.net/ij/index.html) and normalized to β-actin.

IHC analysis

Protein expressions of PCNA, vimentin, and E-cadherin proteins were evaluated by IHC analysis. The details of these antibodies are given Supplementary Table 1. As described earlier, IHC was performed for PCa cancer tissue specimens and xenograft tissues [15]. In brief, tissue specimens were deparaffinized in xylene and rehydrated with an alcohol series, followed by antigen retrieval by boiling slides with citric acid buffer solution (C9999-1000ML, Sigma Aldrich, MO, USA). Subsequently, endogenous peroxidase was blocked by incubating the sections in BLOXALL solution (SP-6000-100, Vector Laboratories, CA, USA) for 10min and washing with TBS. Normal horse serum (S-2012-50, Vector Laboratories, CA, USA) was used to block nonspecific binding, and sections were probed with primary antibodies for PCNA, vimentin, and E-cadherin in normal horse serum and incubated for 1 hr at room temperature. Slides were then washed in TBS with Tween® 20 (TBS-T) and probed with ImmPRESS horseradish peroxidase anti-rabbit IgG (anti-rabbit, #MP-7401, Vector Laboratories, CA, USA) as a secondary antibody. To assess immunoreactivity, ImmPACT DAB (diaminobenzidine, #SK-4105, Vector Laboratories, CA, USA) was used, followed by counterstaining with hematoxylin (Hematoxylin QS, # H-3404, Vector Laboratories, CA, USA).

Fluorescence In situ hybridization (FISH)

To perform FISH, bacterial artificial chromosome (BAC) clones were used to generate a locus-specific probes for TRIP13 (RP11-661C8) and for the chromosome 5 control probe (RP11-91I22). FISH was performed as described earlier using cell lines and PCa samples [14,21]. Briefly, TRIP13 probe and control probe were detected with fluorescein Fab fragments couple to anti-digoxigenin to produce a green color and with Alexa fluor 594 coupled to Streptavidin to generate a red color. Signals were detected with Streptavidin Alexa fluor 594 (S-32356, Invitrogen, CA, USA) and anti-digoxigenin fluorescein Fab fragments (11207741910, Roche, IN, USA). Images were obtained using a CCD camera and ISIS image processing software (Metasystems, Germany).

TRIP13 modulation

siRNA duplexes for TRIP13 were purchased from Dharmacon (CO, USA) and used as previously [14]. Transfections were performed with RNAiMax (Thermo Fisher Scientific, CA, USA). To assess RNA inference, 1 × 10⁵ cells were plated in multi-6-well plates for immunoblot analysis and RNA isolation. After 24 hr, cells were transfected with siRNA duplexes or a non-target control using Oligofectamine (Thermo Fisher Scientific, CA, USA) according to the manufacturer's instructions. Then an identical second transfection was performed after 24 hr, and cells were harvested for RNA and protein isolation after 48 hr of the second transfection. TRIP13 overexpression was performed in RWPE-1 cells using our previously described method using TRIP13 construct [14].

Cell proliferation assay

PCa cells (DU145 and PC3) were plated (2×10⁴ per well) in 12 well plates and were then transfected with a non-target or specific siRNA duplex as described above. TRIP13 knockdown was performed using two independent siRNA-specific duplex sequences targeting TRIP13. The resulting TRIP13 knockdown cells were plated, harvested at selected time points, and counted using a Coulter Z1 particle counter (Beckman Coulter, CA, USA). Untreated cells and cells treated with non-targeting siRNAs served as controls. Each experiment was conducted in triplicate, and results were expressed as means ± SEM for each determination. To evaluate the effect of DCZ0415 (MedChem Express, NJ, USA) on cell proliferation, MTT assays were performed as described earlier [17]. DU145 and PC3 cells were seeded at a density of 10,000 cells/well in 96-well plates and exposed to vehicle (DMSO) or to DCZ0415 (5-20 µM) for 96 hr [15,22]. After treatment, the cells were incubated with MTT reagent for 2 hr, and the formed formazan crystals were dissolved in DMSO, and absorbance was recorded at 540 nm.

Matrigel invasion assay

TRIP13 knockdown cells were used for invasion assays, and cells exposed to a non-target scrambled siRNA were used as controls. Post-transfection (72 hr), cells were plated onto BD BioCoat Matrigel matrices (BD Biosciences, CA, USA) present on the inserts of 24-well culture plates. Medium containing 10% serum was added to the lower chambers to act as a chemoattractant. After 36 hr, the invaded cells were stained with 0.2% crystal violet in methanol, air-dried, and photographed. The inserts were then placed in 10% acetic acid, and absorbance was measured at 560 nm. Invaded cells were also counted.

Colony assay

PCa cells (DU145 and PC3) were cultured in 6-well plates at a density of 1000 cells/well. The cells were treated with vehicle (DMSO) or with DCZ0415 (5-20 µM) for 10 days, and the colonies were stained with crystal violet (0.5%) in 20% methanol for 10 min. After washing several times, the cell colonies were photographed. The effect of DCZ0415 treatment on colony formation was quantified using a 10% acetic acid extraction protocol described earlier [23].

Subcutaneous tumor mouse models for TRIP13 inhibitor treatment

Immunocompromised NSG (NOD/SCID/IL2γ receptor null) mice, 8-10 weeks old, procured from Jackson Laboratory (Farmington, CT, USA) were housed under pathogen‐free conditions in an animal facility. The animal studies were approved and conducted in compliance with a UAB Institutional Animal Care and Use Committee protocol (IACUC-22356). The animals were subcutaneously injected with DU145 (5×10⁶) or PC3 (2×10⁶) cells and were divided into vehicle (n=5 or 6) and DCZ0415 groups (n=5 or 6) when tumor volumes reached ∼100mm³. DCZ0415 was dissolved in DMSO (10%) + PEG300 (40%) + Tween-80 (5%) and saline (45%). On alternate days, the mice were injected intraperitoneally with vehicle or DCZ0415 (50 mg/kg b.wt.). Tumor volumes were measured, and mice were observed for health-related symptoms. The mice were sacrificed upon completion of the experiments, and tumors were harvested, photographed, weighed, and processed for formalin-fixed paraffin-embedded (FFPE) block preparation and hematoxylin and eosin (H&E) and IHC staining.

Toxicity analysis for serum samples from mice

NSG mice (n=3, per group) were treated with vehicle or DCZ0415 (50 mg/kg b.wt.) on alternate days. After treatment blood samples were collected from these mice at weeks 1, 2, and 4, and serum was isolated. Toxicity analysis of serum was performed as described earlier [16].

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5.0. The data are represented as means ± SEM or ± SD. A two-tailed Student t test was conducted to perform comparisons between the groups. P-values ≤ 0.05 were considered statistically significant.

Results

TRIP13 is overexpressed in PCa

RNA-sequence analysis using the Cancer Genome Atlas prostate cancer data showed elevated expressions of TRIP13 in PCa (Fig. 1A). To validate this finding, immunoblot analysis using TRIP13-specific antibody was performed which demonstrated higher TRIP13 protein expression in metastatic PCa tissues relative to benign or primary prostate tissues (Fig. 1B). IHC and immunofluorescence analyses confirmed overexpression of TRIP13 in primary and metastatic PCa tissues (Fig. 1C, 1D). Additionally, FISH analysis revealed a copy number gain at the TRIP13 locus in a case of PCa (Fig. 1E), suggesting a mechanism for TRIP13 overexpression in a subset of tumors.

Fig. 1.

TRIP13 is overexpressed in PCa and is associated with metastasis. (A) TRIP13 mRNA expression in PCa from NGS data. N0- No regional lymph node metastasis, N1- Metastases in 1 to 3 axillary lymph nodes. (B) Immunoblot analysis showing TRIP13 protein expression in bengin, primary, and metastatic PCa tissues. (C, D) IHC and immunofluorescence staining showing expression of TRIP13 in bengin, primary and metastatic PCa tissues. (E) FISH using a TRIP13 locus-specific probe for a PCa sample compared to a normal prostate sample with two copies of TRIP13.

TRIP13 promotes PCa cell proliferation and invasion

Since TRIP13 is implicated as an oncogenic driver in various cancers including PCa [[14], [15], [16],[24], [25], [26], [27], [28]], we investigated its role in PCa cell proliferation and invasion. Using siRNA duplexes targeting TRIP13, we performed knockdown experiments with aggressive PCa cell lines (DU145 and PC3). Knockdown efficiency was confirmed by immunoblots (Fig. 2A, Supplementary Figs.1A-B). Silencing of TRIP13 in DU145 and PC3 cells led to a reduction in PCa cell proliferation compared to non-targeting control cells (Figs. 2B, 2C). We next assessed the effect of TRIP13 knockdown on cell invasion using Boyden chamber Matrigel invasion assays. TRIP13-silenced DU145 and PC3 cells showed less invasion relative to controls (Figs. 2D, 2E). Conversely, ectopic expression of TRIP13 in prostate epithelial RWPE-1 cells showed enhanced invasion compared to LacZ-expressing controls (Fig. 2F). Together, these findings demonstrate that TRIP13 promotes proliferation and invasion of PCa cells.

Fig. 2.

Role of TRIP13 in PCa cell proliferation and invasion. (A) Immunoblot analysis showed the protein expression of TRIP13 in DU145 and PC3 PCa cells treated with either of two specific or independent TRIP13 siRNA duplexes. (B, C) Cell proliferation assay of DU145 and PC3 cells using either of two TRIP13 siRNA duplexes or a non-targeting siRNA control. (D, E) DU145 and PC3 cells were used in the Boyden chamber Matrigel invasion assay, in which TRIP13 was transiently knocked down using either of two independent TRIP13 siRNA duplexes. Non-targeted siRNA-treated cells served as controls. The invaded cells were stained, and the absorbance was read at 560 nm. The bar graphs are shown with ± SEM. (F) Boyden chamber Matrigel invasion assay showing that overexpression of P4HA1 in RWPE-1 cells elevated their invasive potential.

TRIP13 inhibition reduces PCa cell proliferation, induces apoptosis, and modulates EMT markers

To evaluate the therapeutic potential of TRIP13 inhibition, we used DZ0415, a small molecule inhibitor of TRIP13. MTT assays showed that DU145 treatment significantly reduced proliferation of DU145 and PC3 cells in a dose-dependent manner (Fig. 3A). A colony formation assay further demonstrated that DCZ0415 significantly inhibited long-term clonogenic growth of DU145 and PC3 cells (Figs. 3B, 3C). DCZ0415 treatment reduced PCNA expression and increased cleaved caspase 3, indicating induction of apoptosis (Fig. 3D, Supplementary Fig. 1 C-D). Furthermore, the TRIP13 inhibitor modulated EMT markers. In both DU145 and PC3 cells, N-cadherin and vimentin levels were reduced, and E-cadherin was upregulated (Fig. 3E, Supplementary Fig. 1 E-F).

Fig. 3.

DCZ0415 treatment reduces cell growth and colony formation, induces apoptosis, and modulates EMT markers in PCa cells. (A) DU145 and PC3 cells were treated for 96 hr with vehicle (DMSO) or with DCZ0415 (5-20 µM), and cell viability was examined by MTT assays. Error bar indicates mean ± SD, *p < 0.01, **p < 0.001. (B) DU145 and PC3 cells were seeded in six-well plates and treated for 14 days with a vehicle or with DCZ0415 (5-20 µM). The colonies were stained with crystal violet. (C) Quantification was performed by extracting colonies with 10% acetic acid. Error bars indicate means ± SD, **P < 0.001. DU145 and PC3 cells were treated for 48 hr with vehicle (DMSO) or DCZ0415 (5-20 µM), and cell extracts were prepared for immunoblot analysis. (D) Expressions of PCNA, cleaved caspase 3 and TRIP13, and (E) Expressions of N-cadherin, E-cadherin and vimentin in DU145 and PC3 cells treated with vehicle or DCZ0415.

Targeting TRIP13 with DCZ0415 reduces tumor growth

We next evaluated the efficacy of DCZ0415 using DU145 and PC3 xenograft mouse models. When the volume of tumor reached approximately 100 mm³, mice were treated with vehicle control or DCZ0415. Compared to controls, DCZ0415 treatment reduced tumor size, weight, and volume in both DU145 and PC3 xenografts (Figs. 4A-C). IHC staining of the tumor tissues showed lower PCNA expression, consistent with slower tumor growth in DCZ0415-treated mice. Additionally, tumors from the treated group exhibited higher E-cadherin and lower vimentin expression, indicating partial reversal of the EMT (Figs. 4D, 4E). Toxicity evaluation revealed no adverse effects in DCZ0415-treated mice; all parameters remained within normal reference ranges (Supplementary Table 2).

Fig. 4.

DCZ0415 treatment reduces prostate tumor growth and expression of PCNA and modulates EMT markers in NSG mouse models. Mice were subcutaneously injected with DU145 (5×10⁶) or PC3 (2 × 10⁶) cells to initiate tumor growth. When the volume of tumor reached ∼100mm³, the mice were treated with vehicle (control) or with DCZ0415 (50 mg/kg b. wt. on alternate days). (A). Photographs of DU145 and PC3 xenografts after treatment with vehicle or DCZ0415 at the termination of the experiment. (B). Weights of DU145 and PC3 xenografts at the termination of experiments for vehicle- and DCZ0415‐treated mice. Error bar indicates mean± SD, *p<0.001. (C). Growth kinetics of DU145 and PC3 xenografts in mice treated with vehicle or DCZ0415. Error bar indicates mean ± SD,*p < 0.001. (D&E). H&E and IHC staining for PCNA, vimentin, and E-cadherin in DU145 and PC3 xenografts of the vehicle or DCZ0145-treated mice. Scale bar: 20µm.

Discussion

TRIP13 has an oncogenic role and is overexpressed or amplified in various cancers. In the present study, we noted and validated TRIP13 overexpression in both primary and metastatic PCa tissues, consistent with reports linking TRIP13 expression to PCa progression [29]. Additionally, we observed, in a subset of PCa cases, genomic amplification at the TRIP13 locus, suggesting that gene dosage contributes to its elevated expression. These findings align with prior bioinformatics analyses that identified, for various cancers, that TRIP13 is a candidate oncogene and a diagnostic biomarker [30].

Our functional studies demonstrated that TRIP13 knockdown in aggressive PCa cell lines inhibited proliferation and invasion. Conversely, ectopic expression of TRIP13 in prostate epithelial cells enhanced invasive capacity. These findings provide evidence supporting an oncogenic role of TRIP13 in PCa and extend results of earlier reports for other malignancies [16,31]. Mechanistically, TRIP13 may exert these effects by modulating regulators of the EMT, a process necessary for cancer invasion and metastasis. The EMT is characterized by the loss of epithelial markers and gain of mesenchymal markers, facilitating tumor cell dissemination [32,33]. Studies have shown that TRIP13 interact with YWHAZ gene (Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta) and promote cell invasion and EMT process [26,34]. Consistent with this, we observed that TRIP13 inhibition led to a reversal of the EMT, with upregulation of E-cadherin and downregulation of vimentin and N-cadherin.

We also evaluated the therapeutic potential of pharmacologic inhibition of TRIP13 using the small-molecular compound DCZ0415. Treatment with DCZ0415 reduced cell proliferation, induced apoptosis, suppressed clonogenic potential, and modulated EMT markers. DCZ0415 treatment of mice also reduced tumor growth in xenografts without evidence of systemic toxicity, supporting its in vivo efficacy and safety. These findings are consistent with our previous work with colorectal cancer and pancreatic cancer models, in which TRIP13 targeting with DCZ0415 yielded similar antitumor effects [16,17].

In addition to its role in cell proliferation and in the EMT, TRIP13 participates in the DNA damage response and in homologous recombination, suggesting that, when dysregulated, it contributes to genomic instability [14]. Its interaction with spindle checkpoint proteins such as p31^comet, and its involvement in the degradation of HORMA domain proteins during meiosis, highlight its relevance in cell cycle regulation [35]. TRIP13 is also linked to cancer stemness and resistance to chemotherapy and radiation [36,37], showing that targeting TRIP13 may enhance sensitivity to standard-of-care therapies in advanced PCa.

Clinically, TRIP13 overexpression or amplification could serve as a biomarker to identify patients who would benefit from targeted inhibition. Although DCZ0415 is presently in preclinical testing, our findings highlight its potential utility either as monotherapy or in combination with current therapeutic agents such as AR antagonists or DNA damage response inhibitors [38,39]. Given the growing interest in precision oncology, incorporating TRIP13 status into molecular stratification strategies may help personalize treatment for patients with advanced or treatment-resistant PCa.

In summary, this investigation provides evidence that TRIP13 has an oncogenic role in PCa by enhancing cellular proliferation and invasion, and by regulating the EMT. TRIP13 expression is elevated in primary and metastatic PCa samples and may be driven, in part, by genomic amplification. Pharmacologic inhibition of TRIP13 with DCZ0415 suppresses cell proliferation in vitro and tumor growth in vivo, supporting its therapeutic potential.

While our study provides compelling evidence that TRIP13 is involved in promoting PCa and represents a potential therapeutic target, there are some limitations that warrant consideration. First, more in-depth studies are needed to explore the precise mechanisms for inhibition of PCa progression upon targeting TRIP13. Second, the current in vivo findings are based on subcutaneous PCa models that may not fully recapitulate the tumor microenvironment and the complexity of human PCa. Future studies are needed in PCa PDX models expressing TRIP13 to confirm these findings. Third, studies are warranted to evaluate TRIP13 as a predictive biomarker in PCa and investigate the synergy of TRIP13 inhibition with current therapies in AR-dependent and -independent PCa models.

CRediT authorship contribution statement

Farrukh Afaq: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation. Satya S. Pathi: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. Balabhadrapatruni V.S.K. Chakravarthi: Writing – original draft, Methodology, Investigation. Darshan Shimoga Chandrashekar: Writing – original draft, Visualization, Investigation, Data curation. Shannon Carskadon: Methodology. Sameer Al Diffalha: Visualization. Lakshmi P. Kunju: Visualization, Formal analysis. Nallasivam Palanisamy: Visualization, Methodology. Upender Manne: Writing – review & editing, Writing – original draft, Investigation, Funding acquisition, Formal analysis, Conceptualization. Rajesh Singh: Writing – review & editing, Writing – original draft, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Sooryanarayana Varambally: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.