β-catenin signaling in HNSCC cells

Journal Pre-proof Inhibition of CD44 sensitizes cisplatin-resistance and affects Wnt/ β-catenin signaling in HNSCC cells

Souvick Roy, Madhabananda Kar, Shomereeta Roy, Swatishree Padhi, Amit Kumar, Shweta Thakur, Yusuf Akhter, Gianluca Gatto, Birendranath Banerjee PII:

S0141-8130(19)37395-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.131

Reference:

BIOMAC 14435

To appear in:

International Journal of Biological Macromolecules

Received date:

16 September 2019

Revised date:

20 December 2019

Accepted date:

13 January 2020

Please cite this article as: S. Roy, M. Kar, S. Roy, et al., Inhibition of CD44 sensitizes cisplatin-resistance and affects Wnt/β-catenin signaling in HNSCC cells, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2020.01.131

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof Inhibition of CD44 sensitizes cisplatin-resistance and affects Wnt/β-catenin signaling in HNSCC cells

Souvick Roy1, Madhabananda Kar2, Shomereeta Roy1, Swatishree Padhi1, Amit Kumar3, Shweta Thakur4, Yusuf Akhter4,5, Gianluca Gatto3, Birendranath Banerjee1* 1

Molecular Stress and Stem Cell Biology Group, School of Biotechnology, KIIT, Bhubaneswar,

Professor and Head, Department of Surgical Oncology, All India Institute of Medical Sciences

Department of Electrical and Electronic Engineering, University of Cagliari, via Marengo 2,

re

3

-p

(AIIMS), Bhubaneswar, Odisha-751019, India.

lP

09123 Cagliari, Italy. 4

ro

2

of

Odisha-751024, India.

Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central

Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Vidya Vihar,

ur

5

na

University of Himachal Pradesh, Shahpur, Himachal Pradesh 176206, India.

*

Jo

Raebareli Road, Lucknow, Uttar Pradesh 226025, India. Correspondence: Birendranath Banerjee, Associate Professor, KIIT School of Biotechnology,

KIIT University, Bhubaneshwar-751024, Odisha, India: E-mail: [email protected], phone: +91- 9090840042 . Fax: 0674-2378776

Journal Pre-proof Abstract CD44 is one of the key cancer stem-like cell (CSC) marker and may have a potential role in tumorigenesis. In this study, we investigated the role of CD44 in prognosis of HNSCC patients, its possible crosstalk with Wnt/β-catenin signaling and modulating cisplatin resistance. We observed increased expression of CD44 in the cut margin of recurrent HNSCC patients were associated with poor prognosis. We observed that inhibition of CD44 by using 1,2,3,4

of

tetrahydroisoquinoline (THIQ) modulates the expression of Wnt/ β-catenin signaling proteins

ro

and further silencing of β-catenin also decreases the expression of CD44. This led us to

-p

investigate the possible protein-protein interaction between CD44 and β-catenin. Co-

re

immunoprecipitation study illustrated possible interaction between CD44 and β-catenin which

lP

was further confirmed by molecular docking and molecular dynamic (MD) simulation studies. Molecular docking study revealed that one interface amino acid residue Glu642 of β -catenin

na

interacts with Lys92 of CD44 which was also present for 20% of simulation time. Furthermore, we observed that inhibition of CD44 chemosensitizes cisplatin-resistant HNSCC cells towards

ur

cisplatin. In conclusion, this study investigated the possible role of CD44 along with Wnt/ β-

Jo

catenin signaling and their possible therapeutic role to abrogate cisplatin resistance. Key words: CD44; Chemoresistance Wnt/β-catenin signaling

Journal Pre-proof Introduction Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent type of solid tumors in India [1, 2]. This type of cancer primarily originated into the epithelium of the mouth, throat, oral cavity, etc [1]. In recent time there are significant advancements in the field of cancer detection and management, but still, the 5-year survival rate remains poor in HNSCC [3]. The development of local or distant metastasis and resistance towards chemotherapeutic drugs

of

remains a major challenge [4]. In addition, lack of awareness and later presentation of the disease

ro

also contribute towards poor prognosis of HNSCC patients in India [5, 6].

-p

Several reports also suggested that presence of subset of cells in the bulk of the tumor known as

re

cancer stem cells (CSCs) are responsible for aberrant tumor growth, metastasis and intrinsic

lP

therapy resistance [7, 8]. The concept of the presence of CSCs has gained importance in a variety of tumours recently and several strategies were developed to counter these. One of the CSC

na

marker such as OCT4 (octamer-binding transcription factor 4) has been identified as a biomarker

ur

in several cancers including breast, colon, lung and oral cancer [9, 10]. Previous studies also suggested that increased expression of OCT4 is associated with poor prognosis and resistance to

Jo

cisplatin in OSCC [11]. KLF4 another CSC marker is involved wide range of cellular processes such as cell proliferation, apoptosis, migration, inflammation, differentiation and tissue homeostasis [12]. There is a paradox exist regarding the role of KLF4 in different cancers. It acts either as oncogene or tumor suppressor gene in different cancers. In case of GI tract related malignancies such as colon and gastric cancer it acts as tumor suppressor gene while in breast, skin and head and neck cancer increased expression of KLF4 was reported corroborating its oncogenic role [13-16]. One of the most important CSC markers includes CD44, which is a cell surface receptor for hyaluronate and regulates the interaction of cells with substrates and

Journal Pre-proof promote cellular migration [17]. It controls several signaling pathways such as receptor tyrosine kinases (RTKs), including Met and VEGF-2 and G-protein coupled receptors, such as C-X-C chemokine receptor type [18]. In case of HNSCC CD44+ population were demonstrated to have enhanced tumorigenicity as well as resistance towards chemotherapeutic drugs [19]. The role of CD44 in prognosis of different malignant tumors, such as lung cancer, liver cancer, breast cancer along with HNSCC

of

has been reported earlier [20, 21]. Moreover, overexpression of CD44 in tumors has been

ro

associated with therapy resistance and an increased risk of disease relapse in HNSCC [20].

-p

Franzmann et al. has also reported the overexpression of soluble CD44 in oral rinses of HNSCC

re

patients [22]. Previously it was reported that CD44highCD24low cell population from oral cancer

lP

cells have increased CSC population, EMT properties along with increased drug resistance with tumorigenic potential [23]. Thus targeting CD44 can be a promising therapeutic target for

na

HNSCC treatment.

ur

Several strategies has been developed in order to prevent binding of hyaluronate to it receptor

Jo

CD44. Harada et al, reported F-16438s as inhibitors of hyaluronic acid (HA) binding to CD44 [24]. Several studies also reported that hyaluronan oligosaccharides prevent binding of HA to CD44 and inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt signaling pathway [25, 26]. In a previous study it was reported that sulfasalazine selectively induced apoptosis in CD44v-expressing head and neck cancer cells and promoted the sensitivity of tumors to anti-EGFR therapy [27]. Liu et al reported 1,2,3,4 tetrahydroisoquinoline (THIQ) as non-glycosidic inhibitors of CD44-HA [28]. It has been established that Wnt/β-catenin signaling plays an intricate role in maintenance of CSC phenotype in several cancers [29, 30]. Earlier, we reported the role of β-catenin in

Journal Pre-proof mediating cisplatin resistance by regulating the DNA damage repair in HNSCC [1]. GSK3β, one of critical mediator of Wnt/ β-catenin signaling has important role in regulation of self-renewal of CSCs [31]. GSK3β is involved in suppression of Wnt signaling pathway by the degradation of β-catenin through the formation of destruction complex [32]. Thus studying the molecular crosstalk between CD44 and Wnt/β-catenin signaling pathway will provide an additional mechanical insight to understand the CSC mediated therapy resistance in HNSCC. In this study,

of

we aimed to elucidate the role of CD44 in disease relapse and prognosis of HNSCC patients. We

ro

also investigated the role of CD44 along with Wnt/ β-catenin signaling and their possible

-p

therapeutic role to abrogate cisplatin resistance in HNSCC.

re

Materials and Methods

lP

Ethics statement

na

The study was approved by the institutional ethics committee of School of Biotechnology and Kalinga Institute of Medical Sciences (KIMS), KIIT University and conducted according to the

ur

Helsinki declaration. The human sample collection was followed strictly as per institutional

Jo

ethical board guidelines. Informed consent was obtained from all subjects or their nominees prior to participation in the study. Patient sample collection 102 samples from patients operated for HNSCC were collected at the time of surgical removal of the tumor tissue and the cut margin area and stored appropriately. The Cut margin (CM) is adjacent non-tumorous epithelial tissue and during surgery the surgeon usually removes 2-4 cm from the peripheral boundary of the tumor which is visually observed as tumor free normal tissue. It is sometimes supported by a frozen section biopsy and pathologically observed to be

Journal Pre-proof devoid of tumor. Voluntary consent forms were duly signed by patients/guardian before collection of each sample. Drugs, antibodies and primers The drug cisplatin and 1,2,3,4 tetrahydroisoquinoline (THIQ) were obtained from Cipla (Mumbai, India) and Sigma Aldrich (Oakville, Canada) respectively. β-catenin, KLF4, CD44

of

isoform 10, anti-mouse secondary antibody and anti-rabbit secondary antibody were procured

ro

from Abcam (Cambridge, UK). OCT4, GSK3β, p- GSK3β (Ser 9) and GAPDH antibody was procured from Cell Signaling Technologies (Danvers, Massachusetts, USA) and IMGENEX

lP

Maintenance of human HNSCC cell line

re

Technologies (San Deigo, California, USA).

-p

(Bhubaneswar, India) respectively. All the primers were procured from Integrated DNA

na

HNSCC cell line, UPCI-SCC-131 and CAL-27 were cultured in monolayer and maintained in DMEM (GIBCO, Life Technologies, Grand Island, NY, USA) with 1% antibiotic (100 units of

ur

penicillin and 10 mg/ml of streptomycin (HIMEDIA, Mumbai, India), 10% FBS (GIBCO, Life

Jo

Technologies, Grand Island, NY, USA) and 1% (w/v) of L-glutamine (HIMEDIA, Mumbai, India) in a humidified incubator containing 5% CO2 maintained at 37 °C as described previously [1]. UPCI-SCC-131 cell line was derived from a 56 year old male having well differentiated squamous cell carcinoma in floor of mouth and CAL-27 cell line was derived from a 56 year old male having poorly differentiated squamous cell carcinoma in tongue. HNSCC-derived cell lines UPCI-SCC-131 and CAL-27 were generously gifted by Dr. Susanta Roychoudhury (former Scientist of the Indian Institute of Chemical Biology (IICB) CSIR, Govt of India, Kolkata, India)

Journal Pre-proof and Dr. Amrita Suresh (Department of Head and Neck Oncology, Mazumdar Shaw Medical Center, Narayana Health, Bangalore, India) respectively. Generation and maintenance of cisplatin-resistant HNSCC Cell lines Cisplatin-resistant (CisR) variants of each cell line were derived from each parental cell line by intermittent and stepwise exposure to cisplatin. The exposure of the drug to the cells was

of

performed by dose incremental strategy (IC12.5-IC50). After each exposure the surviving fractions

ro

were cultured in drug free medium until it becomes confluent. Each dose of the drug were administered for four cycles and after final cycle of IC50 dose cells were maintained at IC12.5 of

-p

cisplatin and experiments were carried out further.

lP

re

CD44 inhibition in HNSCC cells

Inhibition of CD44 was achieved in HNSCC cells after treatment with 1,2,3,4

na

tetrahydroisoquinoline (THIQ) [28]. HNSCC cells were seeded in 6 well plates at a density of 2 X 105 and allowed to grow till 70% confluence followed by treatment with different

ur

concentrations (1-5 mM) of THIQ for 24 hours.

Jo

siRNA-mediated transient silencing of β-catenin siRNA-mediated transient silencing of SCC-131 and CisR-SCC-131 cells were performed as reported earlier [1]. Briefly, 1X 105 cells were seeded in 60 mm tissue culture dish and allowed to grow till 50% confluence. Subsequent scrambled siRNA and β-catenin siRNA transfections were carried out using DharmaFECT transcription reagent (Dharmacon, Horizon Discovery, Cambridge, UK). The silencing of β-catenin was confirmed by qRT-PCR and Western blotting respectively.

Journal Pre-proof RNA extraction and quantitative real-time PCR Cells and patient tissue samples were used for total RNA extraction by TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) as described previously [1, 2, 33]. Quantitative real-time PCR analyses were performed for β-catenin, other CSC markers (OCT4, KLF4, CD44) with Power Up SYBR Green (2x) (Applied Biosystems, USA). β-actin was used as housekeeping gene and

of

mRNA fold change was calculated by using the 2-ΔΔCT method.

ro

Western blot analysis

-p

Western blot analysis was carried out for HNSCC patient samples and HNSCC cells after different dosage of treatment with THIQ, Cisplatin alone or in combination and in β-catenin

re

silenced HNSCC cells as per previously published protocol [34]. Briefly, cells and patient tissue

lP

samples were harvested and re-suspended in RIPA lysis buffer to obtain protein lysate. Protein

na

estimation was done by using Bradford assay (Bio-rad Laboratories, USA). 50 μg of protein lysates were separated on 12% SDS-PAGE and transferred onto a PVDF membrane (Merck,

ur

Bangalore, India). The membrane was blocked with 5% skimmed milk (HIMEDIA, Mumbai,

Jo

India) in PBST for 1hr at room temperature (RT) and then probed with the primary antibody (1:2000) at 4ºC overnight. The membrane was washed with 0.25% PBST and probed with secondary antibody (1:4000) for 2hrs at RT. The blots were visualized by enhanced chemiluminescence using X-ray film (Kodak, India). Immunohistochemistry analysis Immunohistochemical analysis of HNSCC FFPE tissue blocks was performed as per laboratory established protocol with minor modifications [33, 34]. Briefly, sections were cut into 3-μm sections and dried at 60 °C for 3 h, deparaffinized and dehydrated. Antigen retrieval was done by

Journal Pre-proof the use of microwave method in 10mM Sodium citrate buffer pH 6.0 for 20 minutes. Peroxide block was performed for 30 minutes in dark at RT followed by blocking with with 5% BSA in PBS for and then probed with monoclonal anti-rabbit CD44 primary antibody (1:1000) at 4°C overnight. Next day, sections were washed with PBS and then incubated with the secondary antibody (1:1000) conjugated with HRP for 90 minutes at RT followed by washing and treatment with DAB chromogen for 5 minutes at RT. Sections were washed and counterstained with

of

haematoxylin. Imaging of stained slides was done using Leica (DM 2000) bright-field

ro

microscopy. The expression of the marker was scored by multiplying the percentage of positive

-p

cells (0-100%) with the intensity (weak: 1, moderate: 2 and strong: 3) to obtain a maximum

re

score of 300.

lP

Cell viability assay

Cell viability was checked by MTT assay in CD44 inhibited parental SCC-131 cells, CisR-SCC-

na

131 Cells, parental CAL-27 cells, CisR-CAL- 27 cells, and β-catenin silenced CisR-SCC-131

ur

cells post treatment with different dosage of cisplatin as reported earlier [1]. Cells were seeded in

Jo

96-well plates at a density of 1x104 cells per well and allowed to adhere overnight followed by treatment with cisplatin (1-20 µM) for 24 h. After treatment period MTT reagent [3-(4,5Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)] was added to each well and incubated for 4 hours at 37ºC for formation of formazon crystals. The formazon crystals were dissolved in dissolution solution and absorbance was measured at 570 nm in ELISA reader (Biotek, Germany). Clonogenic cell survival assay Colony-forming capacity was performed by clonogenic assay in HNSCC cells after different dosage of cisplatin treatment as reported earlier [1]. 500 cells/well were seeded in a 6-well cell

Journal Pre-proof culture plate and allowed to adhere overnight followed by treatment with cisplatin (1-10 µM) for 24 hours. After that, medium containing drug was replaced with fresh medium and cells were allowed to form colonies for 7-8 days. Thereafter, the medium was removed and colonies were stained with 0.2% crystal violet prepared in methanol. Then, the wells were washed with distilled water, and colonies were counted using gel documentation system (UVP, Germany).

of

Cell cycle analysis

ro

Cell cycle analysis was performed in HNSCC cells after different dosage of cisplatin treatment as reported earlier [1]. Cells were seeded at a density of 1 X 105 cells in `6 well plate. Cells were

-p

harvested and re-suspended in phosphate-buffered saline (PBS). Cells were subsequently fixed in

re

70% cold ethanol and stored overnight at -20ºC. Post incubation cells were pelleted at 3000 rpm

lP

for 5 minute and subsequently washed with PBS. Followed by incubation with propidium iodide containing RNase A and triton-X-100 and incubated for 20-25 minutes in dark at room

na

temperature. Cell cycle distribution analysis was performed using FACS CANTO II (Becton &

ur

Dickinson, CA, USA) with an event count of 10,000 events per sample. Analysis of data was

cycle.

Jo

done by FACS diva software. Early apoptotic cells were measured in Sub G0 phase of the cell

Wound healing assay

Wound healing assay was performed in HNSCC cells after cisplatin treatment as reported earlier [35]. Briefly, cells were cultured in 6 well tissue-culture plates and allowed to grow till 90% confluence. The wound was provided by using sterile micro tip by scratching. Then cells were treated with cisplatin and allowed to grow for 24 hours. The image of the wound was captured at

Journal Pre-proof 0 hour and 24 hour time interval under and analyzed by Image J software and percentage closure of the wound was calculated. Sphere formation assay CD44 inhibited parental SCC-131 cells, CisR-SCC-131 Cells, parental CAL-27 cells, CisRCAL- 27 cells, and β-catenin silenced CisR-SCC-131 were subjected to sphere formation assay.

of

In a 6 well plate, 500 cells/well were seeded in serum-free DMEM medium supplemented with

ro

Epidermal growth factor (10ng/ml), Basic Fibroblast growth factor (10 ng/ml) and 1% B27 supplement. The medium was added every 2 days and formation of sphere-like structures were

-p

visible after 3-4 days. The images of spheres for each group were captured after 10 days under an

lP

Co-immunoprecipitation (Co-IP) assay

re

inverted microscope (Olympus, Shinjuku, Tokyo, Japan) at 20X magnification.

na

Co-imunoprecipitation assay was performed in the protein lysate of CD44 inhibited SCC-131, CisR-SCC-131 cells and β-catenin silenced SCC-131 and CisR-SCC-131 cells as described

ur

previously [3]. The protein G agarose beads were blocked in 1% BSA overnight at 40c. Protein

Jo

lysate from cells as well as tissues were incubated with anti-CD44-rabbit antibody and anti- βcatenin mouse antibody separately overnight at 40C. The blocked protein G agarose beads were incubated with IgG rabbit (for control) and IgG mouse (for control) for 4 hours at 40c. The protein-antibody solution was incubated with the blocked protein G agarose beads for 4 hours at 40C. Beads were washed thrice with wash buffer. Beads were then suspended in a SDS-sample loading buffer and the soluble proteins were separated by 12% SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti- CD44-rabbit antibody and anti- β-catenin mouse antibody. Protein sequence retrieval and Molecular docking analysis

Journal Pre-proof Amino acid sequences and PDB structures of CD44 (PDB ID: 1POZ, the solution structure of the hyaluronan binding domain of human CD44) and β-catenin (PDB ID: 2Z6H, Crystal Structure of Beta-Catenin Armadillo Repeat Region and its C-terminal domain) were retrieved from the UniProt database and Protein Data Bank. Molecular docking studies were performed to study protein-protein interaction between CD44 and β-catenin using ClusPro 2.0 server as reported earlier [36]. In brief, 25 best-docked

of

complexes were obtained. PyMOL molecular graphics program was used for structure

ro

visualization and interactions were plotted using DIMPLOT program of LIGPLOT software

-p

[37]. Two-dimensional plots generated from DIMPLOT program represents the hydrogen bonds

re

and hydrophobic interactions between the interacting amino acid residues [37]. Further analysis was performed for amino acid residues located at the interacting interfaces of these protein

lP

complexes.

na

Localization of energetic frustration in protein molecules The degree of local energetic frustration in two proteins namely of CD44 (PDB ID: 1POZ) and

ur

β-catenin (PDB ID: 2Z6H) was evaluated by using Frustratometer, a web-based tool

Jo

(http://www.frustratometer.tk) as reported earlier [38] (Ref). In brief, this web-based server employs the structural files or PDB IDs of the query proteins as input and depicts three different regions of frustrations i.e. highly frustrated (red color), minimally frustrated (green color) and neutral regions (gray color). The regions with high local frustrations were considered as biologically important because of its involvement in binding or allostery of other proteins. These regions were further used for analysis. Molecular Dynamics Simulation

Journal Pre-proof The docked complex of β-catenin (PDB ID: 2Z6H) and CD44 (PDB ID: 1POZ) obtained from molecule docking was further subjected to MD simulation. The missing hydrogen atoms were added in complex using psfgen package of VMD software [39]. The protein-protein complex system was solvated in a water box and to obtain a neutral system counter-ions were added. The simulation box edge was [80 104 146], and with water molecules, the system consisted of a total 115200 atoms. TIP3P force-field parameter was used for water molecules [40] and Charmm22

of

force-field parameters for protein atoms. Protein residue protonation state was assigned using

ro

Propka software [41].

-p

Energy minimization was followed by gradual heating of the system to 300 Kelvin (K) in steps

re

of 30 K with positional constraints of 50 kcal mol-1Å-2 on carbon alpha atoms for a simulation

lP

time of 0.3 ns. The constraints were slowly released in steps of 10 kcal mol-1Å-2. Initial equilibration of the system without any constraints was performed for a simulation time length of

na

3 ns followed by molecular dynamics (MD) simulation of 50 ns, performed in NPT ensemble

ur

with T=300K, and 1atm pressure. MD simulations are performed using the NAMD software package [42] and the analysis was done using the VMD software [39]. Further technical details

Jo

have been reported in our previous studies [43-45]. Statistical analysis

Statistical analysis was performed for three independent experiments using the GraphPad Prism 6 software. Student’s t test was performed to assess statistical significance. P < 0.05 or less was considered as statistically significant. Results

Journal Pre-proof Increased expression of CD44 in the cut margin region of recurrent HNSCC patients is associated with poor prognosis We evaluated the association of expression of CD44 in HNSCC by meta-analysis, using publicly available Oncomine datasets [46]. Three HNSCC datasets reported by Ginos et al., Peng et al., and Cromer et al. were used for dataset analysis [47-49]. The meta-analysis revealed that higher expression of CD44 in tumor specimens as compared to normal mucosa samples (Supplementary

of

Figure 1A). We further performed Kaplan Meier analysis for overall survival of HNSCC patients

ro

based on CD44 expression status by using TCGA dataset. It was observed that increased

-p

expression of CD44 was associated with poor prognosis in HNSCC patients (Supplementary

re

Figure 1B).

lP

In this study, 102 HNSCC patients were included and segregated into two cohorts namely recurrent and non-recurrent. Western blot (WB) and gene expression analysis were performed to

na

check the expression of CD44 for each HNSCC patients in surgically removed tumor tissue and

ur

their respective cut margin counterparts. WB and gene expression analysis revealed that

Jo

recurrent HNSCC patients exhibited increased expression of CD44 in cut margin (CM) region as compared to their tumor counterpart. In the case of non-recurrent HNSCC patients, no significant difference in expression of CD44 was observed between CM and tumor region (Figure 1A-B and C). Immunohistochemical analysis also showed increased expression of CD44 in CM of recurrent HNSCC patients as compared to the tumor region. In contrast, strong membranous expression of CD44 was observed in tumor tissues of non-recurrent HNSCC patients (Figure 2AB). In order to evaluate the association of expression of CD44 with different clinicopathological factors, Chi-square test was performed (Table 1). Chi-square with different clinicopathological

Journal Pre-proof factors revealed a significant correlation between disease recurrence and the expression of CD44 (Table 1). Kaplan Meier (KM) analysis showed no significant differences in disease-free survival (DFS) and overall survival (OS) of 102 HNSCC patients based on high expression levels of CD44 in the cut margin or tumor region. Furthermore, it was observed that recurrent HNSCC patients having higher expression of CD44 in cut margin region had reduced DFS and OS as compared to those with high expression of CD44 in the tumor region (Figure 3A-B).

ro

of

1,2,3,4 tetrahydroisoquinoline (THIQ) inhibits expression of CD44 in HNSCC cell lines We next investigated the effect of 1,2,3,4 tetrahydroisoquinoline (THIQ) on the expression of

-p

CD44. Previously, it was reported that THIQ treatment resulted in inhibition of CD44-

re

hyaluronan mediated signaling pathway [28]. The HNSCC cell lines were treated with different

lP

concentrations of THIQ (1, 2 and 5mM). WB and gene expression analysis revealed that 1mM THIQ treatment resulted in decrease in the expression of CD44 in parental HNSCC cells as well

na

as their respective cisplatin-resistant counterparts (Figure 4A and C). We further performed flow

ur

cytometric analysis of percentage CD44 positive population after treatment with 1 mM THIQ in

Jo

parental and cisplatin-resistant HNSCC cells. It was observed that 1mM THIQ treatment decreased the percentage of CD44 positive population in parental as well as in cisplatin-resistant HNSCC cells (Figure 4B).

Inhibition of CD44 modulates the expression of Wnt/β-catenin signaling proteins and CSC markers We further investigated the effect of CD44 inhibition on the expression of Wnt/β-catenin signaling proteins and CSC markers (OCT4, KLF4) in HNSCC cells. It was observed that 1mM THIQ treatment also resulted in decreased expression of β-catenin and decreased

Journal Pre-proof phosphorylation of GSK3β (Ser 9). In contrast, the expression of GSK3β was increased. Inhibition of CD44 also decreases the expression of CSC markers (OCT4, KLF4) in HNSCC cells (Figure 5A-B). Silencing of β-catenin decreases the expression of CD44 and other CSC markers In order to evaluate the role of β-catenin in modulating the expression of CSC markers, we

of

transfected the HNSCC cells with siRNA. The silencing efficiency was confirmed by Western

ro

blot and gene expression analysis. We also investigated the gene and protein expression of CSC markers after silencing of β-catenin in HNSCC cells. It was observed that silencing of β-catenin

re

increased expression of GSK3β (Figure 6A-B).

-p

led to a decrease in the expression of CD44 and other CSC markers (OCT4, KLF4) along with

lP

Presence of protein-protein interaction between CD44 and β-catenin in HNSCC cells

na

From previous observations, we found that inhibition of CD44 regulates Wnt/β-catenin signaling and silencing of β-catenin decreased the expression of CD44 and other CSC markers. Thus we

ur

evaluated the protein-protein interaction, if any between CD44 and β-catenin by co-

Jo

immunoprecipitation study. Co-immunoprecipitation study demonstrated the loss of expression of β–catenin in CD44 inhibited CisR-SCC-131 cells post pull down with CD44 and vice versa. Besides, silencing of β–catenin in CisR-SCC-131 cells also exhibited loss of expression of CD44 post pull down with β–catenin and vice versa (Figure 7A). We further elucidate protein-protein interaction among β-catenin and CD44 by in silico analysis. Molecular docking study identified several amino acid residues located at the interacting interfaces of these two proteins. The docked models of the protein-protein complex were represented as cartoon structures (Figure 7B) and their interacting binding partners are

Journal Pre-proof highlighted in enlarged surface view. The interactive amino acid residues between β-catenin (PDB ID: 2Z6H) and CD44 (PDB ID: 1POZ) at the binding interface were represented in Supplementary Figure 2 and Supplementary Table 1. We further performed Frustratometer analysis to quantify and localize the distribution of local energetic frustrations occurring within the proteins. In this study, we have identified a couple of highly frustrated regions (shown in red color) often termed as disordered regions that might be

of

involved in protein-protein interactions at a molecular and cellular level and are shown in

ro

Supplementary Table 2 and Figure 7C . In this study, we found that one interface amino acid

-p

residue Glu642 of β -catenin interacts with Lys92 of CD44 (Supplementary Table 3). These

re

results demonstrated that CD44 and β–catenin indeed interacts with each other.

lP

Molecular Dynamics (MD) simulation analysis of CD44-β–catenin protein-protein

na

complexes

The stability of the protein-protein complex was evaluated from the root mean square deviation

ur

calculation on the carbon-alpha atoms. The hydrogen bond (H-bond) interactions between the

Jo

CD44 – β-catenin protein-protein complex was evaluated during the simulation. We found six persistent H-bond interactions across the protein-protein interface (Figure 7D). H-bond interaction His135 – Arg549 and Glu6 – Arg550 were present for more than 50 % of simulation time, while the interaction Lys92 –GluE642 revealed from frustatometer analysis was present for 20 % of simulation time. The interaction energy between the residues of CD44 protein and β-catenin protein was calculated by evaluating the non-bonded energy values comprising of Van der Waals and electrostatic energy, using the energy plugin of NAMD software. We obtained the average

Journal Pre-proof interaction energy value of -236 ± 67 kcal/mol and noted that key residues contributing up to 75% of interaction energy (Figure 7E). To further investigate the role of interface residues in protein-protein interactions, we performed three single point in-silico mutations for β-catenin involving residues R549, R550 and E642, and compare the results with wild type simulation. In general for the three mutant β-catenin proteins, we found different interaction partner residue of CD44 residue involved in H-bonded interactions

of

at the interface (Figure 7F). With an exception for mutant R550A β-catenin–CD44 complex, the

ro

other mutant protein-protein complexes displayed a lower number of persistent H-bonded

-p

interactions across the interface. Interestingly, the impact of β-catenin mutant (R550A) on the

re

protein-protein complex was evident from a richer interaction network at the interface. The

lP

R550A mutation in β-catenin resulted in much more persistence nature of H-bond interactions, such as Gln11 – Gln548, His135 – Arg549, Lys92 –GluE642, with respect to the wild type β-

na

catenin – CD44 complex and other mutant complexes (Figure 7F).

ur

Inhibition of CD44 chemosensitizes HNSCC cells towards cisplatin

Jo

Next, we investigated whether inhibition of CD44 by THIQ treatment has any effect on the chemosensitivity of HNSCC cells towards cisplatin. First, we evaluated cell viability in CD44 inhibited HNSCC cells after treatment with different concentrations of cisplatin (1-20 µM). It was observed that cisplatin treatment resulted in a decrease in cell viability of CD44 inhibited Parental as well as cisplatin-resistant HNSCC cells in dose dependent manner (Figure 8A). The clonogenic assay was also performed to check colony-forming capacity and cell survival in CD44 inhibited HNSCC cells post cisplatin treatment. We found that cisplatin treatment resulted in decreased colony-forming ability and cell survival in CD44 inhibited HNSCC cells (Figure 8B-C). In addition, we assessed the cell cycle distribution of CD44 inhibited HNSCC cells after

Journal Pre-proof treatment with 1 µM of cisplatin. It was observed that cisplatin treatment resulted in increased G2/M phase arrest of cisplatin-resistant HNSCC cells as compared to their parental counterparts (Figure 8D). We also investigated the effect of CD44 inhibition on cellular migration of HNSCC cells post cisplatin treatment by in vitro scratch assay. It was observed that inhibition of CD44 in HNSCC cells resulted in dose-dependent decrease in percentage wound closure post cisplatin treatment (1-10 µM) (Figure 8E). Besides, we investigated the sphere-forming capacity of CD44

of

inhibited HNSCC cells. It was observed that inhibition of CD44 with THIQ treatment resulted in

ro

decreased sphere-forming capacity of parental as well as cisplatin-resistant HNSCC cells (Figure

-p

8F). These results suggested that inhibition of CD44 chemosensitizes HNSCC cells towards

re

cisplatin.

lP

Discussion

The incidence rate of HNSCC in India is about one third among all other cancers [1, 6]. Lifestyle

na

habits such as cigarette smoking, gutkha chewing and alcohol consumption further contribute to

ur

disease progression [5] . The recurrence rate and local or distant metastasis were also remaining

Jo

quite high in advanced cases [1, 50]. The presence of CSCs in the bulk of the tumor has been reported to be involved in disease relapse and therapy resistance in HNSCC by decreasing the apoptosis induced by chemotherapeutic drugs or radiotherapy [51] . CD44, one of the key CSCs marker has been linked with the prognosis of HNSCC patients depending on its expression level [52]. Cancer cells having CD44 positive have been linked with increased chemoresistance towards cisplatin, increased migration potential in different cancers [19, 52]. Wnt/β-catenin signaling pathway one of the significant pathway which governs the maintenance of CSC phenotype [53] . CD44 was previously reported as a Wnt target gene [54], but there was no conclusive evidence of protein-protein interaction between CD44 and β-catenin in cancer. In this

Journal Pre-proof study, we elucidated the role of CD44 in disease relapse and prognosis of HNSCC patients. We also investigated the effect of inhibition of CD44 on expression of Wnt/β-catenin signaling proteins and other cancer stem cell markers. The protein-protein interaction between CD44 and β-catenin were explored by co-immunoprecipitation study and obtained results were further validated by molecular docking and MD-simulation analysis. Furthermore, we also evaluated the effect of CD44 inhibition on chemosensitivity of HNSCC cells towards cisplatin.

of

In our previous study, we observed that increased expression of OCT4, KLF4 and β-catenin in

ro

the cut margin area of recurrent HNSCC patients which was associated with poor prognosis [1,

-p

16]. In the current study, we observed increased expression of CD44 in the cut margin region of

re

recurrent patients as compared to their tumor counterpart suggesting the presence of stem like

lP

tumor cells in the area. This finding is correlating with disease proliferation towards periphery of the tumor which may gain pluripotency and stemness adaptability [51]. Previously it was

na

reported that genetic alterations facilitate the progression of cancer from pre-malignant lesions which may not result in morphological changes [55]. Thus, the concept of “molecular surgical

ur

margin” incorporating molecular characteristics into surgical margin analysis may give a more

Jo

accurate assessment of the cells at cut margin and also help in prognostication. Chi-square analysis also revealed significant association between high expression of CD44 in cut margin region and disease recurrence. Kaplan Meier analysis showed that recurrent patients having high expression of CD44 in the cut margin region had poor prognosis. These observations guided us to hypothesize the role of CD44 in disease relapse and prognosis of HNSCC patients depending on its expression level. The presence of CSCs in the bulk of the tumor is associated with therapeutic resistance and disease relapse. In order to evaluate the role of CD44 in chemoresistance HNSCC cells were

Journal Pre-proof treated with THIQ which was previously reported as CD44 inhibitor [28]. First, the optimum concentration of THIQ treatment to inhibit CD44 was evaluated by WB and gene expression analysis. We observed that 1mM THIQ treatment resulted in a significant decrease in CD44 expression in Cisplatin-resistant HNSCC cells as well as in their parental counterparts. Flow cytometric analysis also showed that 1mM THIQ treatment decreased the % CD44 positive population.

of

As reported earlier CD44 as a Wnt signaling pathway target gene [54] we further evaluated the

ro

effect of CD44 inhibition in expression of different proteins involved in Wnt/ β-catenin signaling

-p

pathway along with the expression of other CSC markers such as OCT4 and KLF4. It was

re

observed that inhibition of CD44 resulted in a decreased expression of β-catenin and

lP

phosphorylation of GSK3β (Ser 9) along with decreased expression of other CSC markers. In contrast the expression of GSK3β, which is a key suppressor of β-catenin was increased. Thus it

na

can be speculated that inhibition of CD44 might regulate the expression of essential proteins involved in Wnt/β-catenin signaling pathway. These observations were in accordance with

ur

previous studies that reported the role Wnt/β-catenin pathway as one of the critical regulator for

Jo

maintenance of CSC phenotype in different cancers [53, 54]. We also investigated the expression of CSC markers in parental as well as CisR-SCC-131 cells after the silencing of β-catenin. It was observed that silencing of β-catenin led to decreased expression of CSC markers in parental as well as CisR-SCC-131 cells. In our previous study we observed the potential role of β-catenin in promoting cisplatin resistance in HNSCC cells [1]. In this study we observed that siRNA mediated silencing of β-catenin also chemosensitizes cisplatin-resistant HNSCC cells (Supplementary Figure 3).

Journal Pre-proof Co-immunoprecipitation study illustrated that loss of expression or inhibition of either of these two proteins resulted in decreased expression of the other one. This preliminary results suggested a possible protein-protein interaction between CD44 and β-catenin. We further carried out in silico studies based on the existing literature and previous experimental data, to substantiate the interactions between CD44 and β-catenin. It was observed that one interface amino acid residue of Glu642 of β-catenin interacts with Lys92 of CD44. The interacting amino acids residues

of

observed during Frustratometer analysis, were lying within the same interacting regions as

ro

obtained from docking study and dimplot analysis also suggested a strong protein-protein

-p

interaction between these two proteins.

re

MD simulation analysis of CD44-β-catenin protein-protein complex further confirmed the results

lP

obtained from molecular docking study. We observed stable protein-protein complex of CD44β-catenin, which was evident from six persistent H-bond interactions across the protein-protein

na

interface. The interaction of Lys92 of CD44 and Glu642 of β-catenin obtained from frustatometer analysis was also present for 20% of simulation time. It was also observed that that

ur

interacting residues of CD44 and β-catenin protein contributes towards 75% of interaction

Jo

energy of the CD44-β-catenin protein-protein complex. Thus, supporting the role of the interface residues in providing stable CD44 protein and β-catenin protein complex. Furthermore, in-silico mutations for β-catenin involving residues R549, R550 and E642 exhibited different interaction partner residue of CD44 residue involved in H-bonded interactions at the interface. It was observed that, mutant R550A β-catenin–CD44 complex exhibited more abundant interaction network at the protein interfaces as evident from more persistent nature of H-bond interactions as compared to the wild type and other mutant complexes. In detail for the same mutation (R550A), we note a much more persistence nature of H-bond interactions, such as

Journal Pre-proof Gln11 – Gln548, His135 – Arg549, Lys92 –GluE642, with respect to the wild type β-catenin – CD44 complex. This observation is consistent with better protein-protein interaction energy value calculated for the R550A mutant protein complex simulation (Figure 4F), with respect to the wild type and other mutant complexes. Since CD44 is required for the maintenance of CSC phenotype, we inhibited CD44 in HNSCC cells and evaluated the chemoresistance or chemosensitivity of HNSCC cells towards cisplatin. It

of

was observed that inhibition of CD44 by THIQ treatment chemosensitizes HNSCC cells towards

ro

cisplatin as evident from MTT assay, clonogenic assay and sphere-forming assay. Cell cycle

-p

analysis of HNSCC cells resulted in increased G2/M phase arrest of cisplatin-resistant HNSCC

re

cells as compared to parental counterpart. The increased G2/M phase arrest was associated with

lP

increased apoptosis and it can be speculated that CD44 inhibition promoted the G2/M phase arrest in cisplatin-resistant HNSCC cells. Furthermore, wound healing assay also revealed that

na

THIQ treatment resulted in decrease migration potential in parental as well as cisplatin-resistant

ur

HNSCC cells post cisplatin treatment. Thus it can be concluded that inhibition of CD44 promoted chemosensitivity towards cisplatin in HNSCC cells.

Jo

In conclusion our study reiterated the fact that CD44 inhibition affects Wnt/β-catenin signaling and there by affects the maintenance of CSC phenotype. Thus, CD44 can be used as a potential target for therapeutic interventions in HNSCC to overcome cisplatin induced therapeutic resistance. Conflict of Interest

Journal Pre-proof The authors declare that part of this study was presented in NCRI Cancer Conference, 2018 by Mr. Souvick Roy. The work was not published anywhere in any form, the abstract submitted for the NCRI conference has been displayed in online catalogue of NCRI conference website. Acknowledgement The authors acknowledge grant from Virtual National Oral Cancer Institute [Understanding the

of

Disease Biology and Epigenetic Diversity of Oral Cancer in India: (Implications for New

ro

Diagnostics and Therapeutics)], Department of Biotechnology, Government of India (Grant NoBT/PR17576/MED/30/1690/2016). YA lab is funded by Indian Council of Medical Research

-p

(Government of India) and Department of Biotechnology (Government of India). We sincerely

re

thank Dr. Susanta Roychoudhury, former Scientist of Indian Institute of Chemical Biology

lP

(IICB), Kolkata, India and Dr. Amrita Suresh, Department of Head and Neck Oncology, Mazumdar Shaw Medical Center, Narayana Health, Bangalore, India for generously providing us

Jo

References

ur

na

HNSCC cell line UPCI-SCC-131 and CAL-27 respectively.

[1] Roy S, Kar M, Roy S, Saha A, Padhi S, Banerjee B. Role of beta-catenin in cisplatin resistance, relapse and prognosis of head and neck squamous cell carcinoma. Cellular oncology (Dordrecht). 2017. [2] Roy S, Roy S, Kar M, Padhi S, Saha A, Anuja K, et al. Role of p38 MAPK in disease relapse and therapeutic resistance by maintenance of Cancer Stem Cells (CSCs) in Head and Neck Squamous Cell Carcinoma. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2018. [3] Roy S, Roy S, Kar M, Thakur S, Akhter Y, Kumar A, et al. p38 MAPK pathway and its interaction with TRF2 in cisplatin induced chemotherapeutic response in head and neck cancer. Oncogenesis. 2018;7:53. [4] Sayed SI, Dwivedi RC, Katna R, Garg A, Pathak KA, Nutting CM, et al. Implications of understanding cancer stem cell (CSC) biology in head and neck squamous cell cancer. Oral oncology. 2011;47:237-43.

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[5] Joshi P, Dutta S, Chaturvedi P, Nair S. Head and neck cancers in developing countries. Rambam Maimonides medical journal. 2014;5:e0009. [6] Shah SB, Sharma S, D’Cruz AK. Head and neck oncology: The Indian scenario. South Asian Journal of Cancer. 2016;5:104-5. [7] Fanali C, Lucchetti D, Farina M, Corbi M, Cufino V, Cittadini A, et al. Cancer stem cells in colorectal cancer from pathogenesis to therapy: Controversies and perspectives. World Journal of Gastroenterology : WJG. 2014;20:923-42. [8] Gangemi R, Paleari L, Orengo AM, Cesario A, Chessa L, Ferrini S, et al. Cancer stem cells: a new paradigm for understanding tumor growth and progression and drug resistance. Current medicinal chemistry. 2009;16:1688-703. [9] Tsai LL, Hu FW, Lee SS, Yu CH, Yu CC, Chang YC. Oct4 mediates tumor initiating properties in oral squamous cell carcinomas through the regulation of epithelial-mesenchymal transition. PloS one. 2014;9:e87207. [10] Li SJ, Huang J, Zhou XD, Zhang WB, Lai YT, Che GW. Clinicopathological and prognostic significance of Oct-4 expression in patients with non-small cell lung cancer: a systematic review and meta-analysis. Journal of thoracic disease. 2016;8:1587-600. [11] Tsai LL, Yu CC, Chang YC, Yu CH, Chou MY. Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2011;40:621-8. [12] Yu F, Li J, Chen H, Fu J, Ray S, Huang S, et al. Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene. 2011;30:2161-72. [13] Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC, et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer research. 2005;65:2746-54. [14] Choi BJ, Cho YG, Song JW, Kim CJ, Kim SY, Nam SW, et al. Altered expression of the KLF4 in colorectal cancers. Pathology, research and practice. 2006;202:585-9. [15] Pandya AY, Talley LI, Frost AR, Fitzgerald TJ, Trivedi V, Chakravarthy M, et al. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2004;10:2709-19. [16] Roy S, Kar M, Roy S, Padhi S, Saha A, Banerjee B. KLF4 expression in the surgical cut margin is associated with disease relapse of Oral squamous cell carcinoma. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology. 2019. [17] Senbanjo LT, Chellaiah MA. CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells. Frontiers in Cell and Developmental Biology. 2017;5:18. [18] Fuchs K, Hippe A, Schmaus A, Homey B, Sleeman JP, Orian-Rousseau V. Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44. Cell death & disease. 2013;4:e819. [19] Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:973-8.

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[20] Spiegelberg D, Kuku G, Selvaraju R, Nestor M. Characterization of CD44 variant expression in head and neck squamous cell carcinomas. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35:2053-62. [21] Xu H, Tian Y, Yuan X, Wu H, Liu Q, Pestell RG, et al. The role of CD44 in epithelial– mesenchymal transition and cancer development. OncoTargets and therapy. 2015;8:3783-92. [22] Franzmann EJ, Reategui EP, Pereira LH, Pedroso F, Joseph D, Allen GO, et al. Salivary protein and solCD44 levels as a potential screening tool for early detection of head and neck squamous cell carcinoma. Head & neck. 2012;34:687-95. [23] Ghuwalewala S, Ghatak D, Das P, Dey S, Sarkar S, Alam N, et al. CD44(high)CD24(low) molecular signature determines the Cancer Stem Cell and EMT phenotype in Oral Squamous Cell Carcinoma. Stem cell research. 2016;16:405-17. [24] Harada H, Nakata T, Hirota-Takahata Y, Tanaka I, Nakajima M, Takahashi M. F-16438s, novel binding inhibitors of CD44 and hyaluronic acid. I. Establishment of an assay method and biological activity. The Journal of antibiotics. 2006;59:770-6. [25] Ghatak S, Misra S, Toole BP. Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway. The Journal of biological chemistry. 2002;277:38013-20. [26] Lu X, Huang X. Design and syntheses of hyaluronan oligosaccharide conjugates as inhibitors of CD44-Hyaluronan binding. Glycoconjugate journal. 2015;32:549-56. [27] Yoshikawa M, Tsuchihashi K, Ishimoto T, Yae T, Motohara T, Sugihara E, et al. xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma. Cancer research. 2013;73:1855-66. [28] Liu L-K, Finzel BC. Fragment-Based Identification of an Inducible Binding Site on Cell Surface Receptor CD44 for the Design of Protein–Carbohydrate Interaction Inhibitors. Journal of Medicinal Chemistry. 2014;57:2714-25. [29] de Sousa EMF, Vermeulen L. Wnt Signaling in Cancer Stem Cell Biology. Cancers. 2016;8. [30] Liu F, Millar SE. Wnt/beta-catenin signaling in oral tissue development and disease. Journal of dental research. 2010;89:318-30. [31] Shigeishi H, Biddle A, Gammon L, Emich H, Rodini CO, Gemenetzidis E, et al. Maintenance of stem cell self-renewal in head and neck cancers requires actions of GSK3beta influenced by CD44 and RHAMM. Stem cells (Dayton, Ohio). 2013;31:2073-83. [32] Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S, Kikuchi A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. The EMBO journal. 1998;17:1371-84. [33] Padhi SS, Roy S, Kar M, Saha A, Roy S, Adhya A, et al. Role of CDKN2A/p16 expression in the prognostication of oral squamous cell carcinoma. Oral oncology.73:27-35. [34] Padhi S, Saha A, Kar M, Ghosh C, Adhya A, Baisakh M, et al. Clinico-Pathological Correlation of beta-Catenin and Telomere Dysfunction in Head and Neck Squamous Cell Carcinoma Patients. Journal of Cancer. 2015;6:192-202. [35] Saha A, Shree Padhi S, Roy S, Banerjee B. Method of detecting new cancer stem cell-like enrichment in development front assay (DFA). Journal of stem cells. 2014;9:235-42. [36] Comeau SR, Gatchell DW, Vajda S, Camacho CJ. ClusPro: a fully automated algorithm for protein-protein docking. Nucleic acids research. 2004;32:W96-9. [37] Wallace AC, Laskowski RA, Thornton JM. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein engineering. 1995;8:127-34.

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[38] Jenik M, Parra RG, Radusky LG, Turjanski A, Wolynes PG, Ferreiro DU. Protein frustratometer: a tool to localize energetic frustration in protein molecules. Nucleic acids research. 2012;40:W348-51. [39] Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. Journal of Molecular Graphics. 1996;14:33-8. [40] Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79:92635. [41] Rostkowski M, Olsson MH, Sondergaard CR, Jensen JH. Graphical analysis of pHdependent properties of proteins predicted using PROPKA. BMC structural biology. 2011;11:6. [42] Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al. Scalable molecular dynamics with NAMD. Journal of computational chemistry. 2005;26:1781-802. [43] Kumar A, Chakravarty H, Bal NC, Balaraju T, Jena N, Misra G, et al. Identification of calcium binding sites on calsequestrin 1 and their implications for polymerization. Molecular bioSystems. 2013;9:1949-57. [44] Kumar A, Delogu F. Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor. Scientific reports. 2017;7:42496. [45] Kumar A, Sechi LA, Caboni P, Marrosu MG, Atzori L, Pieroni E. Dynamical insights into the differential characteristics of Mycobacterium avium subsp. paratuberculosis peptide binding to HLA-DRB1 proteins associated with multiple sclerosis. New Journal of Chemistry. 2015;39:1355-66. [46] Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Varambally R, Yu J, Briggs BB, et al. Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia (New York, NY). 2007;9:166-80. [47] Peng CH, Liao CT, Peng SC, Chen YJ, Cheng AJ, Juang JL, et al. A novel molecular signature identified by systems genetics approach predicts prognosis in oral squamous cell carcinoma. PloS one. 2011;6:e23452. [48] Ginos MA, Page GP, Michalowicz BS, Patel KJ, Volker SE, Pambuccian SE, et al. Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck. Cancer research. 2004;64:55-63. [49] Cromer A, Carles A, Millon R, Ganguli G, Chalmel F, Lemaire F, et al. Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis. Oncogene. 2004;23:2484-98. [50] Padhi SS, Roy S, Kar M, Saha A, Roy S, Adhya A, et al. Role of CDKN2A/p16 expression in the prognostication of oral squamous cell carcinoma. Oral oncology. 2017;73:27-35. [51] Mitra A, Mishra L, Li S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget. 2015;6:10697-711. [52] Baillie R, Tan ST, Itinteang T. Cancer Stem Cells in Oral Cavity Squamous Cell Carcinoma: A Review. Frontiers in Oncology. 2017;7:112. [53] Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461-73. [54] Kim J-H, Park S-Y, Jun Y, Kim J-Y, Nam J-S. Roles of Wnt Target Genes in the Journey of Cancer Stem Cells. International Journal of Molecular Sciences. 2017;18:1604. [55] Westra WH, Sidransky D. Phenotypic and genotypic disparity in premalignant lesions: of calm water and crocodiles. Journal of the National Cancer Institute. 1998;90:1500-1.

Journal Pre-proof Figure Legends Figure 1: Differential expression of CD44 in cut margin and tumor of HNSCC patients. (A) Representative images of western blots showing differential expression of CD44 in cut margin (CM) and Tumor areas of non-recurrent and recurrent HNSCC patients. (B) Quantitative representation of CD44 protein expression in CM and Tumor areas of non-recurrent and recurrent patients. (C) Graphical representation of gene expression of CD44 in CM and Tumor

ro

of

areas of recurrent and non-recurrent HNSCC patients.

Figure 2: Immunohistochemical analysis of CD44 in cut margin and tumor of HNSCC

-p

patients. (A) Representative images of immunohistochemical analysis of CD44 in CM and

re

tumor of recurrent and non-recurrent patients HNSCC patients. (B) Graphical representation of

lP

mean Histoscore of CD44 expression in CM and Tumor areas of recurrent and non-recurrent

na

patients HNSCC patients.

Figure 3: High expression of CD44 in cut margin of recurrent HNSCC patient was

ur

associated with poor prognosis. (A-B) Kaplan-Meier curves of disease-free and overall survival

Jo

of 102 HNSCC patients based on expression of CD44 in CM and tumor. (C-D) Kaplan-Meier curves of disease-free and overall survival of recurrent HNSCC patients based on expression of CD44 in CM and tumor.

Figure 4: 1,2,3,4 tetrahydroisoquinoline (THIQ) inhibits the expression of CD44 in HNSCC cells. (A and C) Western blot (WB) and gene expression analysis of CD44 in parental and cisplatin-resistant HNSCC cells after treatment with different concentrations of THIQ. (B) Flow cytometric analysis of CD44 positive population in parental and cisplatin-resistant HNSCC cells after treatment with 1mM THIQ. The data represented is the mean ± SD of three independent

Journal Pre-proof experiments. P value < 0.05 or less was considered as statistically significant. (*p < 0.05), (**p < 0.005), (*** p < 0.001). Figure 5: THIQ mediated inhibition of CD44 modulates the expression of Wnt/ β-catenin signaling proteins and CSC markers. (A)WB analysis of Wnt/β-catenin signaling proteins and CSC markers after inhibition of CD44 by THIQ. (B) Gene expression analysis of β-catenin, KLF4 and OCT4 after inhibition of CD44 by THIQ treatment in HNSCC cells. The data

of

represented is the mean ± SD of three independent experiments. P value < 0.05 or less was

ro

considered as statistically significant. (*p < 0.05), (**p < 0.005), (*** p < 0.001).

-p

Figure 6: siRNA mediated silencing of β-catenin decreases the expression of CD44 and CSC

re

markers in HNSCC cells. (A-B) WB and gene expression analysis of β-catenin, GSK3β and

lP

CSC markers after siRNA mediated silencing of β-catenin. The data represented is the mean ± SD of three independent experiments. P value < 0.05 or less was considered as statistically

na

significant. (*p < 0.05), (**p < 0.005), (*** p < 0.001).

ur

Figure 7: Presence of protein-protein interaction between CD44 and β-catenin in HNSCC

Jo

cells. (A) Co-immunopreciptation study to evaluate protein-protein interaction between CD44 and β-catenin after inhibition of CD44 and silencing of β-catenin in parental and cisplatinresistant HNSCC cells. (B) The docked model of protein-protein complex of CD44 and βcatenin. (C) Frustratometer analysis to quantify and localize the distribution of local energetic frustrations occurring within the CD44 and β-catenin proteins. (D) Hydrogen bonded interaction between CD44 –β-catenin protein-protein complex (E) CD44–β-catenin interaction energy plot for the wild type and mutant complexes. The interaction energy corresponds to the non-bonded energy values comprising of Van der Waals and electrostatic energy. (F) Hydrogen bonded interaction between CD44-β-catenin wild type and mutant protein-protein complexes.

Journal Pre-proof Figure 8: THIQ mediated inhibition of CD44 chemosensitizes HNSCC cells towards cisplatin. (A) Percentage cell viability after treatment with different concentrations of cisplatin (1–20 μM) in CD44 inhibited HNSCC cells by THIQ. (B-C) Percentage cell survival and colony forming capacity in CD44 inhibited parental and cisplatin-resistant HNSCC cells (SCC-131 and CAL-27) after cisplatin treatment (1–10 μM). (D) Cell cycle profile analysis of CD44 inhibited parental and cisplatin-resistant HNSCC cells (SCC-131 and CAL-27) after treatment with 1 µM

of

of cisplatin. (E) Graphical representation of percentages of wound closure after 24 hours

ro

cisplatin treatment (1-10 µM) in CD44 inhibited parental and cisplatin-resistant HNSCC cells.

-p

(F) Sphere forming capacity of parental and cisplatin-resistant HNSCC cells after THIQ

re

treatment. The data represented is the mean ± SD of three independent experiments. P value < 0.05 or less was considered as statistically significant. (*p < 0.05), (**p < 0.005), (*** p <

lP

0.001).

Jo

ur

na

Table 1: Association of expression of CD44 and clinicopathological characteristics in HNSCC patients (n=102).

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

Authors Statement S.R. performed the in vitro studies, conducted data analysis, validated the data in patient samples and wrote the manuscript. M.K. performed the surgery, provided the clinical samples and interpreted the patient data. S.H.R performed the Western Blot analysis and Immunoprecipitation study. S.P performed the IHC and analyzed the data. A.K. and G.G. performed the MD simulations study and interpreted the data S.T. and Y.A. conducted the in silico analysis and interpreted the bioinformatics data. B.N.B. conceived the idea and guided through the experiments, wrote and edited the manuscript. All authors reviewed the manuscript.

Journal Pre-proof

Table 1: Association of expression of CD44 and clinicopathological characteristics in HNSCC patients (n=102)

Clinicopathological characteristics

No. of patients

Age ≥60 <60

High Expression of CD44 Cut margin

Tumor

34 21

27 20

P value

0.654 61 41

0.609 9 46

53 49

29 26

Site of tumor Tongue Buccal Mucosa

36 66

Recurrence Non-Recurrent Recurrent

73 29

6 41

12 35

32 23

41 6

72 30

42 13

30 17

Lymphovascular Invasion (LVI) No Yes

95 7

52 3

43 4

Perineural Invasion (PNI) No Yes

75 27

41 14

34 13

Bone metastasis No Yes

91 11

48 7

43 4

Jo

ur

na

lP

re

24 31

Lymph node metastasis No Yes

0.867

24 23

-p

ro

15 87

of

Gender Female Male Histological Grade Moderate Well

0.056

0.001(*)

0.166

0.543

0.801

0.494

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12