Abstract 2579: Kras expression level regulates heparanase activity

Abstract

Abstract Introduction: Sarcomas are locally and systemically aggressive, significantly interacting with extracellular matrix. The purpose of this study is to quantify the expression of heparanase by K-ras expressing sarcoma cells. Methods: Balb/c3T3 and K234 (Ki-ras-transformed Balb/c 3T3) cells (ATCC) were cultured using DMEM with 5% FBS. Heparanase assays were performed with the heparan degrading enzyme assay kit (cat# MK412; Takara) on cell lysate for each cell line according manufacturer protocol. Dhmaracon on-Target Plus SMARTpool siRNA was used to knock down Kras. Total RNA was isolated with Qiagen RNeasy kit, cDNA was made using Invitrogen SuperScript™ III First-Strand Synthesis System, real time RT-PCR was done by use SYBR green Master Mix and Applied Biosystems® 7500 system, mRNA level for HPSE and PPBP was measured by RTPCR. The UCSC genome browser was used to show HPSE sequence and primer location. Results: The heparan degrading activity of lysate from K234 cells was 20-30 times higher than from Balb/c3T3 cells. However, nine pairs of primers for mHPSE RT-PCR results show that mHPSE mRNA is expressed differently in Balb/c3T3 and K234 cell with K234 cell mHPSE mRNA level is lower than in Balb/c 3T3 cells. The mRNA sequence is the same in these two cell lines, however. Using siRNA knock down of greater than 80% Kras from K234 and Balb/c3T3 cells, the heparanase activity of lysate decreased by 78% in K234 cells, and 25% in Balb/c3T3 cells. Kras knock down also changed cell morphology in both cell lines, and changed the mRNA levels of HPSE, PPBP and some EMT markers. Conclusion: Kras overexpressed in K234 cells is associated with increase in heparanase activity, and knock down of Kras decreases heparanase activity. However, heparanase activity does not correlate with HPSE (heparanase 1) expression level, so there is likely another mechanism(s) for Kras expression to regulate heparanase activity. Citation Format: Donghong Ju, Mary A. Kosir. Kras expression level regulates heparanase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2579.