Fig. 4

Rhamm-loss results in reduced expression of interferon alpha and gamma pathway components. A The transcriptome of primary MMTV-PyMT tumors was analyzed using RNA-seq as described in the Methods. Analysis of genes that are differentially expressed in Wildtype versus Rhamm^−/− primary tumors using Metascape and GSEA, identified interferon signaling as the top down-regulated pathway in Rhamm^−/− primary tumors. B GSEA Hallmark gene set analyses identified 51 IFNG and IFNA regulated genes as top hallmark gene sets that are down-regulated by Rhamm-loss. Twenty-six of these genes are significantly (p < 0.001) co-expressed with RHAMM in 2 datasets of breast cancer patients (METABRIC and TCGA Pan-Cancer Atlas, c-Bioportal.org) and are shown with Spearman correlation coefficients from METABRIC data sets in the histogram. C STAT1 is an example of an interferon pathway gene that is linearly co-expressed with RHAMM in luminal B breast cancer subtype. Graph is derived from the TCGA Pan-Cancer Atlas data set. D STAT1 protein levels in Wildtype and Rhamm^−/− primary mammary tumors were analyzed by Western blot. Wildtype tumors express significantly more STAT1 protein compared to Rhamm^−/− tumors. Values are the Mean and SEM of n = 3 tumors. *p < 0.05. E STAT1 staining of Wildtype and Rhamm^−/− primary tumors and lung metastases. Wildtype tumors contain more STAT1-positive cells in tumors and the tumor microenvironment than Rhamm^−/− counterparts. F Rhamm mRNA expression was knocked down by siRNA in the Py8119 MMTV-PyMT mammary tumor cell line as described in Methods, and the effect on RHAMM and STAT1 protein expression was quantified using Western blots. Rhamm knock-down strongly reduces STAT1 protein expression