https://openworks.mdanderson.org/sumexp23/1096/thumbnail.jp

Fagbola, Jayden Oluwadamini

Kurie, Jonathan M.

Tan, Xiaochao

English

OpenWorks @ MD Anderson

Evaluation of Manganese (Mn) Binding Proteins in Lung Carcinoma CellsJayden Fagbola1, Xiaochao Tan1, Jonathan M. Kurie1Department of Thoracic/Head & Neck Medical Oncology; University of Texas MD Anderson Cancer Center, Houston, TX1Despite significant improvements in lung cancertherapies in recent years, lung cancer remains asthe leading cause of cancer-related death due toits inclination to metastasize. Recent findingshighlight the metastatic capacity of tumor cells asa result of overexpression of the Golgi integralmembrane protein 4 (GOLIM4), which isfrequently amplified in many human cancers,including lung squamous cell carcinoma (LUSC).GOLIM4 forms a complex with ATPase SecretoryPathway Ca2+ transporting 1 (ATP2C1) on thetrans-Golgi and promotes pro-metastatic vesicletrafficking. Highly expressed GOLIM4 drives lungcancer growth and metastasis. Manganese (Mn)is an essential element that is present in tinyamounts in the human body. Mn treatmentcauses GOLIM4 degradation and inhibits thegrowth of chromosome 3q-amplified LUSC cells.To identify other factors that may mediate theantitumor functions of Mn, we performed a pull-down assay and identified several Mn bindingproteins. In this project, we assessed thebiological roles of these Mn binding proteinsusing several functional assays and aimed todetermine whether these proteins are functionaltargets of Mn treatment.Fig. 1 A. Schematic flow of Mn pull-down assay.B. A volcano graph showing identified Mn bindingproteins. Newly identified Mn binding proteins areindicated in red. We chose four proteins (BLMH,S100A8, SERPINB8, TIMP3) for further functionalstudies based on the peptide abundance.Fig. 2 H1299 cells were transfected with two differentsiRNAs against BLMH, S100A8, SERPINB8, TIMP3, orcontrol siRNA (siCTL), and the mRNA levels of each genewere examined by quantitative real time PCR. Both siRNAscould effectively knock down the target genes.Fig. 3 H1299 cells were transfected with indicatedsiRNAs, and cell proliferation was determined byWST-1 assay. The results show that the twosiRNAs exhibit similar activity in suppressingcancer cell proliferation. All these four genes arerequired for the proliferation of H1299 cancer cells.Fig. 4 H1299 cells were transfected with indicated siRNAsand cell migration was assessed using Boyden chambertranswell assay. The migrated cells were stained andcounted. A. Images of migrated cells. B. Quantification ofmigrated cells. The results were normalized to siCTL. Allthese four proteins were required for the migration of H1299cancer cells, although the effect of siTIMP3 is marginal.Fig. 5 H1299 and H520 cells weretreated with (100 or 200 μM) orwithout Mn for 24 h, and theprotein levels of BLMH, TIMP3,SERPINB8, and GOLIM4 proteinswere determined by Western Blot.We found that Mn treatmentsignificantly reduced the proteinlevel of GOLIM4 but not the otherthree proteins, indicating that theseproteins bind to Mn but are notdegraded by Mn.We identified several significant Mn bindingproteins (BLMH, S100A8, SERPINB8, TIMP3) ina Mn pull-down assay. We used specific siRNAsto knock down these proteins and assess cellproliferation using the WST-1 method and cellmigration using the Boyden chamber trans-wellassay. In addition, we examined the levels ofthese proteins following Mn treatment byperforming a Western blot to investigate if Mncaused degradation of these proteins.A BWe have shown that GOLIM4 is a Mn bindingprotein which is degraded upon Mn exposure.We found that Mn treatment effectivelysuppressed the growth of chromosome 3q-amplified lung cancer cells. We will examine thefunctions of these Mn binding proteins. Wehypothesize that Mn functions by targetingseveral oncogenic proteins.We found that these Mn binding proteins playcritical roles in cell proliferation andmigration. However, Mn treatment did notresult in degradation of these proteins,suggesting that they may not be primary Mntargets in chromosome 3q-amplified lungcancer cells. Thus, we conclude thatGOLIM4, but not other Mn binding proteins,are responsible for Mn-induced cancer cellgrowth inhibition. In the future, we will focuson the functions of GOLIM4 in lung cancergrowth and metastasis.This project is supported by the1R25CA240137-01A1 UPWARDSTraining Program (UnderrepresentedMinorities Working Towards ResearchDiversity in Science) and CPRITResearch Training Award CPRIT TrainingProgram (RP210028).I would like to give a special thank you tomy mentor, Dr. Xiaochao Tan, foreducating and assisting me for theduration of this program.A1) https://lcfamerica.org/2) Matthew B. Schabath, Michele L. Cote;Cancer Progress and Priorities: LungCancer. Cancer Epidemiol BiomarkersPrev 1 October 2019; 28 (10): 1563–1579.3) Tan X, Banerjee P, Guo HF, Ireland S,Pankova D, Ahn YH, Nikolaidis IM, Liu X,Zhao Y, Xue Y, Burns AR, Roybal J, GibbonsDL, Zal T, Creighton CJ, Ungar D, Wang Y,Kurie JM. Epithelial-to-mesenchymaltransition drives a pro-metastatic Golgicompaction process through scaffoldingprotein PAQR11. J Clin Invest. 2017 Jan3;127(1):117-131.IntroductionHypothesisMethodsResults(Bleomycin Hydrolase) is a cytoplasmicpeptidase involved in the inactivation ofglycopeptide bleomycin.(S100 Calcium Binding Protein A8) is acalcium binding protein involved in cell cycleprogression and differentiation.(Serpin Family B Member 8) is an enzymeproduced by platelets and can bind to andinhibit the function of furin.(TIMP Metallopeptidase Inhibitor 3) is aprotein coding gene involved in degradationof the extracellular matrix (ECM).BLMHSERPINB8TIMP3S100A8BConclusion & Future Directions Acknowledgements References** P < 0.01*** P < 0.001**** P < 0.0001Binding Proteins

Evaluation of Manganese (Mn) Binding Proteins in Lung Carcinoma Cells

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Evaluation of Manganese (Mn) Binding Proteins in Lung Carcinoma Cells

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