Hsp70 supports the expression of extracellular matrix genes upregulated in human breast cancer

BENJAMIN LANG; SHREYA VENKATESH; DESHENG WENG; AYESHA MURSHID; MARTÍN E. GUERRERO; PATRICK MAGAHIS; KRIS HOLTON; THIAGO BORGES; ANH NHU; STUART K. CALDERWOOD; TAKANORI EGUCHI; JIANLIN GONG

Virginia

Conferencia; IXth International Symposium on Heat Shock Proteins in Biology and Medicine; 2018

Cell Stress Society International

The stress-inducible Hsp70 (Hspa1a/Hspa1b) is clinically associated with breast cancer progression and has been shown to support tumor growth in vivo (1, 2). A molecular basis for these relationships has been suggested by studies that identified HSP70 to support oncogenic signaling, inhibit apoptosis, and overcome senescence (1, 3, 4).Previously our laboratory found Muc1.Tg MMTV-PyMT (MMT) Hsp70-/- mice to exhibit delayed tumor growth, but also exhibit remarkable protection to spontaneous mammary tumor metastases that are characteristic of the MMT mammary tumor model (2,5). We hypothesized that Hsp70 may regulate metastatic processes beyond its above mentioned tumorigenic activities. Our study therefore seeks to determine the mechanistic basis for Hsp70 modulation of mammary tumor metastasis. Our objective is focused towards two research questions:- What genes are most affected by Hsp70 perturbation within metastatic mammary tumors?- How does HSP70 regulate the levels of these mRNAs?Materials and methodsWe utilized an RNA-seq approach to quantitatively identify tumor signaling systems most affected upon Hsp70 perturbation in murine mammary tumors. RNA was extracted from age-matched MMTV-PyMT (MMT) WT and Hsp70-/- mammary tumors from which cDNA libraries were generated. The libraries were sequenced on an Illumina® HiSeq 2500 platform to give approximately 15-20 million reads per sample. Reads were checked for quality using FastQC and aligned to annotated mouse genes (UCSC mm10) using TopHat2. HTSeq-counts were then used as input for differential gene expression (DGE) analysis. DGE analysis was performed in R using the Bioconductor package edgeR (7). EdgeR uses TMM normalized read counts and fisher?s exact test with Benjamini-Hochberg correction for multiple testing to identify significantly altered genes with a false discovery rate (FDR) less than 0.05 (6). Chemical inhibition of Hsp70 in vitro, qPCR, and Western blot, we will then be used to determine how Hsp70 regulates mRNAs of interest.Conclusions- Hsp70 regulates a series of ECM-encoding genesincluding Col1a1, Col1a2, Col5a1, Sparc, Bgn- COL1A1, COL1A2, COL5A1, SPARC and BGN are significantly upregulated in human breast tumor tissue compared to non-tumor tissue- The Hsp70 inhibitor JG98 inhibits Collagen I protein and mRNA levels- As altered ECM composition influences mammary tumor progression, the regulation of ECM mRNAs by Hsp70 may contribute to its role in mammary metastasis- Our findings point to a potential role for Hsp70 in fibrogenic signaling and possible involvement of co-chaperones