CMTM6 drives cisplatin resistance in OSCC by regulating AKT mediated Wnt signaling

Chemoresistance is one of the important factors for treatment failure in OSCC, which can culminate in progressive tumor growth and metastatic spread. Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance, which can be achieved by identifying the causative factors for acquired chemoresistance. In this study, to explore the key cisplatin resistance triggering factors, we performed global proteomic profiling of OSCC lines representing with sensitive, early and late cisplatin-resistant patterns. The top ranked up-regulated protein appeared to be CMTM6. We found CMTM6 to be elevated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analyses of OSCC patient samples indicate that CMTM6 expression is upregulated in chemotherapy-non-responder tumors as compared to chemotherapy-naïve tumors. Stable knockdown of CMTM6 restores cisplatin-mediated cell death in chemoresistant OSCC lines. Similarly, upon CMTM6 overexpression in CMTM6KD lines, the cisplatin resistant phenotype was efficiently rescued. Mechanistically, it was found that CMTM6 interacts with membrane bound Enolase-1 and stabilized its expression, which in turn activates the AKT-GSK3β mediated Wnt signaling. CMTM6 triggers the translocation of β-catenin into the nucleus, which elevates the Wnt target pro-survival genes like Cyclin D, c-Myc and CD44. Moreover, incubation with lithium chloride, a Wnt signaling activator, efficiently rescued the chemoresistant phenotype in CMTM6KD OSCC lines. In a patient-derived cell xenograft model of chemoresistant OSCC, knock-down of CMTM6 restores cisplatin induced cell death and results in significant reduction of tumor burden. CMTM6 has recently been identified as a stabilizer of PD-L1 and henceforth it facilitates immune evasion by tumor cells. Herewith for the first time, we uncovered another novel role of CMTM6 as one of the major driver of cisplatin resistance.


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Head and neck cancer is the sixth most common cancer worldwide with approximately 53,260 73 new cases are being reported in United States alone 1 . Almost 90% of HNSCC cancer cases are 74 Oral squamous cell carcinoma (OSCC) which has emerged as the most common cancer in 75 developing countries. In India, 80000 new OSCC cases are reported in each year with a 76 mortality of ~46000 2 . OSCC patients commonly present with locally advanced (stage III or IV) 77 disease. The treatment modalities of advanced OSCC are surgical removal of primary tumor 78 followed by chemo-radiotherapy 3 . However, neoadjuvant chemotherapy is commonly 79 prescribed for surgically unresectable OSCC tumors that provide more surgical options 4 . In spite 80 of having all these treatment modalities, the 5-year survival rate of advanced tongue OSCC 81 remains less than 50%, which indicates the development of resistance against existing therapy.

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Chemoresistance is one of the major factors for treatment failure in OSCC. The common 83 chemotherapy regimens for OSCC are Cisplatin alone or with 5FU and Docetaxel (TPF) 5 . The 84 tumor shows initial positive response to chemotherapy, but later it acquires chemoresistance, and 85 patient experience relapse with onset of metastatic diseases. The chemoresistant properties could 86 be attributed to enhanced cancer stem cell population, decreased drug accumulation, reduced 87 drug-target interaction, reduced apoptotic response and enhanced autophagic activities 6 . These 88 hallmarks present the endpoint events, when cancer cell had already acquired chemoresistance. 89 Few attempts have been made to understand the molecular mechanism of chemoresistance in shRNA based human kinome study elucidates that microtubule-associated serine/threonine 95 kinase 1 (MAST1) is a major driver of cisplatin resistance in HNSCC. MAST1 inhibitor 96 lestaurtinib, efficiently sensitized chemoresistant cells to cisplatin. Overall the study suggests 97 that MAST1 is a viable target to overcome cisplatin resistance 8 . 98 Here, to elucidate the causative factors those responsible for acquired chemoresistance, we have 19). Approved procedures were followed for patient recruitment and after receiving written 117 informed consent from each patient, tissues samples were collected.  all three time points with appropriate technical and biological replicates were used in an isobaric 134 tag for relative and absolute quantification (iTRAQ) experiment (Fig. 1B). Equal amount of 135 proteins (100 μg) from all samples were taken for tryptic protein preparation following 136 manufacturer's instructions (AB Sciex, USA). Study samples with the tag details used for 137 labelling in iTRAQ experiment are presented in Fig. 1B. Trypsin treatment was performed using 138 trypsin supplied by the manufacturer and incubating at 37 o C for 16-20 hrs. Tryptic peptides were 139 dried at 40 o C using SpeedVac (LabConco, USA). Dried tryptic peptides were dissolved using 140 dissolution buffer and isobaric tags reconstituted with isopropanol were added to be incubated at 141 room temperature for 2 h. After the completion of the reaction, tagged tryptic peptides from all 142 samples were pooled and dried. Tagged tryptic peptides (~250 μg) were subjected to strong 143 cation exchange fractionation using a hand-held ICAT® Cartridge-cation-exchange system 144 (Applied Biosystems, USA). Peptides were eluted using a gradient 30, 50, 80, 120, 250, 300, 400 145 and 500 mM of ammonium formate solutions with a flow rate of 10 drops/min. Each SCX 146 fraction was dried at 40 o C using a Speed Vac (CentriVap, Labconco, USA) and cleaned using 147 Pierce C18 spin column (ThermoFisher Scientific Inc., USA).

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Each SCX fraction was resuspended in 20 μl of buffer (water with 0.1% formic acid) and 149 introduced to easy-nanoLC 1000 HPLC system (Thermo Fisher Scientific, Waltham, MA) 150 connected to hybrid Orbitrap Velos Pro Mass Spectrometer (Thermo Fisher Scientific, Waltham, 151 MA). The nano-LC system contains the Acclaim PepMap100 C18 column (75 µm × 2 cm) 152 packed with 3 μm C18 resin connected to Acclaim PepMap100 C18 column (50 µm × 15 cm) 153 packed with 2 μm C18 beads. A 120 min gradient of 5% to 90% buffer B (0.1% formic acid in 154 95% Acetonitrile) and Buffer A (0.1% formic acid in 5% Acetonitrile) was applied for separation 155 of the peptide with a flow-rate of 300 nl/min. The eluted peptides were electrosprayed with a 156 spray voltage of 1.5 kV in positive ion mode. Mass spectrometry data acquisition was carried out 157 using a data-dependent mode to switch between MS1 and MS2.        to isolate total RNA as per manufacturer's instruction and quantified by Nanodrop. c-DNA was 231 synthesised by reverse transcription PCR using Verso cDNA synthesis kit (ThermoFisher 232 Scientific, Cat # AB1453A) from 300 ng of RNA. qRT-PCR was carried out using SYBR Green 233 master mix (Thermo Fisher scientific Cat # 4367659). GAPDH was used as a loading control.

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The primer (oloigos) details used for qRT-PCR in this article are listed in supplementary table   (TPF). Based on qRT-PCR and immunohistochemistry data, higher abundance of CMTM6 was 325 observed in tumor tissues isolated from non-responders to drug naive OSCC patients (Fig. 1I-K). 326 Similarly, we determined the expression of CMTM6 in drug-naive and post chemotherapy 327 treated paired tumor samples not responding to treatment. The post chemotherapy treated tumor 328 samples showed higher expression of CMTM6 (Fig. 1L). Overall, it was found that CMTM6 329 expression is significantly elevated in chemoresistant lines.

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Targeting CMTM6 reverses cisplatin resistance in squamous cell carcinomas: To delineate 331 the potential role of CMTM6 as a major driver of cisplatin resistance, we generated stable 332 CMTM6 knock down clones in chemoresistant lines using a lentivirus based ShRNA approach.

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Stable clones generated by both the ShRNA showed efficient knock down of CMTM6 ( Fig. 2A).

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Our cell viability and cell death assay data suggests that knock down of CMTM6 significantly (targets 5'UTR of CMTM6 mRNA )followed by treatment with cisplatin (Fig. 3A). The MTT 347 assay and annexin-V/7AAD staining data suggests that ectopic overexpression of CMTM6 in 348 CMTM6KD drug resistant cells results in rescuing the cisplatin resistant phenotype (Fig. 3B, C).

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Similarly, transient overexpression of CMTM6 decreases the expression of γ-H2AX in CMTM6 350 KD cells (Fig. 3D). GSK-3β (S9) expression (Fig. 4C). Earlier, it is reported in the literature that Enolase-1 can 363 activate AKT signaling 13 . In a mass spectrometry based analysis by Burr et al., 2017 mentioned 364 that CMTM6 interacts with membrane enolase-1 14 . Henceforth, we wanted to observe whether 365 CMTM6 interacts with Enolase-1. Our co-immunoprecipitation and confocal microscopy assay 366 confirmed that CMTM6 and Enolase-1 interact with each other and they co-localized in plasma 367 membrane (Fig. 4D, E). Further, we analyzed the expression of Enolase-1 in cytoplasmic and  The immunoblotting and immunostaining data indicates that when CMTM6 is knocked down in 375 chemoresistant lines, there is significant downregulation of β-catenin, p-β-catenin (s552) and 376 non-phospho active β-catenin (Fig. 5A, B). It is important to mention here that AKT is known to 377 phosphorylates β-catenin at s552 and this results in translocation of β-catenin from cytoplasm to 378 nucleus 15 . When Wnt activator lithium chloride (LiCl) was treated with CMTM6KD cells, we 379 observed up regulation of β-catenin and c-Myc expression in chemoresistant cells (Fig. 5C).

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Similarly, MTT assay data suggests the reversal of chemoresistant phenotype of CMTM6KD 381 cells followed by treatment with Wnt activator lithium chloride (LiCl) (Fig. 5D). Further, we  CMTM6ShRNA group but not in NtShRNA group (Fig. 7 A-C). We also observed significantly 418 decreased cell proliferation in cisplatin treated CMTM6ShRNA tumors with reduced expression 419 of Wnt target pro survival genes (Fig. 7D). 24. Guan X, Zhang C, Zhao J, Sun G, Song Q, Jia W. CMTM6 overexpression is associated with 568 molecular and clinical characteristics of malignancy and predicts poor prognosis in gliomas. 569 EBioMedicine 2018