https://openworks.mdanderson.org/sumexp21/1206/thumbnail.jp

Calin, George A., MD, PhD

Rausseo, Paola C.

Sahnoune, Iman

English

OpenWorks @ MD Anderson

INTRODUCTIONGastric cancer is a frequent cause ofdeath worldwide1. It has been linked tovarious environmental factors that differaround the world. In the United States,risk factors vary from smoking to eatingsalty to heavily processed foods, to the H.pylori bacteria. Gastric carcinomas areoften therapy resistant and due to theircellular heterogeneity, this makes itdifficult to establish a “one size fits all”therapeutic approach in patients.MiRNAs are small non-coding RNAs thatfound to be implicated in gene expressionand regulation. One miRNA in particular,miR-10b, has been found to be highlyoncogenic on metastatic cancers, with itsoverexpression linked to proliferation,migration, and tumor size. OncogenicmiRNAs operate via targeting tumorsuppressor genes, silencing expressionand permitting abnormal cells to divideuncontrollably, contributing to the growthof cancerous tumors. Increased levels ofmiR-10b are associated with pooreroverall survival and serve as acontributing prognostic factor2.The limited treatment options aroundgastric cancer has led Calin’s group todevelop a novel therapeutic binding miR-10b. This study reports on the effect ofCompound 25, a derivative of the FDA-approved therapeutic linifanib. Thiscompound was developed incollaboration with the Institute for AppliedCancer Sciences (IACS) at MD AndersonCancer Center.RESULTSAfter treating the three cell lines withCompound 25, we used microscopy toassess morphological and phenotypicchanges within the SNU-1 and AGScells (gastric cancer cell lines).Fortunately, we were able to see thatthere were minimal differences to theGES-1 cell line (normal epithelialgastric line) validating that there waslow miR-10b levels in normal cells,confirming it as a targeted therapeutic.By evaluating RNA expression usingPCR, we tested miR-10b expression inboth cell lines and patient samplesderived from a collaborator at MDAnderson Cancer Center,supplementing our cancer cell line data.We identified a reduction of miR-10bafter the treatment with Compound 25specifically in malignant cells,correlated with the death of malignantcells but not of normal cells.CONCLUSIONOncogenic miR-10b is upregulated ingastric cancer cell lines and haslimited to undetectable expression ina normal gastric line. Compound 25target miR-10b in cancer cell linesand has the potential for translationalapplication in a deadly type ofcancer, the gastric cancer.1. Rawla, P. & Barsouk, A. Epidemiology ofgastric cancer: global trends, risk factorsand prevention. Prz Gastroenterol 14, 26-38, doi:10.5114/pg.2018.80001 (2019).2. Sheedy, P. & Medarova, Z. Thefundamental role of miR-10b in metastaticcancer. Am J Cancer Res 8, 1674-1688(2018).*Diagrams generated in BioRenderMETHODSThe learning step: to gain a strong foundation on my project, I reviewed over 50 papers onmiRNAs and miR-10b in PubMed. After my first week in the laboratory, I was able to perform cellculture and by the second week, I was maintaining several cell lines across three tissue types. Ishadowed senior colleagues on RNA extraction, cDNA preparation, Western blots, and PCRtechniques. I then learned to plate cells, use the microscope, and start new cell lines. By thesixth week, I did my own PCR to generate data for an ongoing publication. Afterwards, I wastaught to evaluate and analyze the data.A Novel Small Molecule Targeting Oncogenic miR-10b in Gastric CancerPaola Rausseo1, Iman Sahnoune1, George A. Calin11Department of Translational Molecular Pathology, MD Anderson Cancer Center, Houston, TXHYPOTHESIS We hypothesize that miR-10b drivescancer through binding tumor suppressorgenes and that Compound 25 decreasesbinding of miR-10b. Therefore, high miR-10b expression in gastric cancer cells willbe impacted by the compound, leading tomalignant cell death.DMSO Compound 25DMSO Compound 2524hrs48hrsGES - 1 AGSDMSO Compound 25SNU-1Our experiments began by plating cells into wells and inserting a sequential dosage ofCompound 25 to find our optimal dosage. For best results, the cells were incubated for up to 48hours. Pictures were taken at 24 and 48 hours at 4x to compare cell density and morphologicaldifferences between the control wells and the wells treated with 5µM of Compound 25. After 48hours, we performed RNA extraction, cDNA preparation and conducted a miR-10b quantitativePCR.Figure 1: Schematic representation of the cell culture procedure using cell lines AGS, SNU-1, and GES-1.Figure 2: Process of treatment for cells with Compound 25. After treatment we waited 48 hours then began the PCR process including RNA extraction and cDNA preparation. Figure 3: Pictures of cells at 24 to 48 hours after treatment with Compound 25. Each condition is represented, with control versus 5µm to show the differences between the cells after drug treatment at two time points. FUTURE DIRECTIONSMore research will be conductedregarding the specific mechanism ofmiR-10b in gastric cancer metastasesand the specific genes that Compound25 is targeting.The experimental step: For our study we cultured three different cell lines. GES-1 is thehuman gastric epithelial cell line of normal origin. AGS is an adherent stomach cancer tissuecell derived from a 54-year-old female. SNU-1 is a metastatic suspension stomach cancertissue cell from a 44-year-old male. These cells have been cultured, passaged and split every 2-3 days.REFERENCES

A Novel Small Molecule Targeting Oncogenic miR-10b in Gastric Cancer

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1206&context=sumexp21

A Novel Small Molecule Targeting Oncogenic miR-10b in Gastric Cancer

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