Nanda, R. K. | bioRxiv Science

bioRxiv2020

Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using glucose

13C based Isotopic tracers of the media components can be used to kinetically track their contribution in the cell systems. A tracer ([U-13C6] glucose) was used to monitor its contribution into the central carbon metabolic pathways of human lung carcinoma (A549) cells by Gas chromatography-mass spectrometry (GC-MS) based mass isotopomer analysis. Calculated average 13C of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), protein acid hydrolysates (serine: 4.51{+/-}0.21 %, glycine: 2.44{+/-}0.31 %, alanine: 24.56{+/-}0.59 %, glutamate: 8.81{+/-}0.85 %, proline: 6.96{+/-}0.53 % and aspartate: 10.72{+/-}0.95 %) and the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), showed significant contribution of 13C glucose. We observed the Warburg effect with higher levels of 13C lactate in the culture filtrate. 13C glycerol levels in culture supernatant showed significant increase with time and amino acids of glucogenic origin also contributed cellular protein biomass. The workflow adopted in this study for 13C analysis could be useful for the metabolic phenotyping of other mammalian cell systems under normal and perturbed (cancer and infection) conditions.

biochemistry↗

bioRxiv2020

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-naive 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-GSK3{beta} mediated Wnt signaling. CMTM6 triggers the translocation of {beta}-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.

cancer biology↗