Supplementary Materials and Methods, Supplementary Table S1. List of 9 PRLR gene isoforms in the UCSC Genome Browser based on RNASeqV2 data. Supplementary Table S2. Protein expression profile by RPPA analysis in Ishikawa tumors treated with mannitol or G129R. Supplementary Table S3. Pathological staining results for PRLR using 2 scoring metrics in normal uterine tissue (n=3 patients) and UCEC-EEA (n=3 patients). Supplementary Figure S1 (supporting Figure 1). Expression of PRLR_SF (uc003jjl.3) in UCEC-EEA patients. Supplementary Figure S2 (supporting Figure 1). Expression of PRLR in human uterine cancer cell lines with both PTENmut and PTENWT backgrounds. Supplementary Figure S3 (supporting Figure 2). Representative images from the Ishikawa model showed the different sizes of primary tumors at the uterine horn in different groups. Supplementary Figure S4 (supporting Figure 3). In vitro efficacy of G129R in Ishikawa and Hec1-A cells under hormone-depleted condition. Supplementary Figure S5 (supporting Figure 3). G129R reduced the viabilities of Ishikawa and Hec1A cells in comparison with PRL stimulation at hormone-depleted condition. Supplementary Figure S6 (supporting Figure 5). siRNAs knock down the expression of FOXO3a or EIF-4EBP1 in Ishikawa and Hec1A cells. Supplementary Figure S7 (supporting Figure 4C & 4D, Supplementary Figure S2, and Supplementary Figure S6). Uncropped western blots produced in this study.
Funding
Department of Defense Ovarian Cancer Research Program
NIH
Deutsche Forschungsgemeinschaft
OCRA
Donald Payne and Michael Redman
ARTICLE ABSTRACT
Abnormal activity of human prolactin (PRL) and its membrane-associated receptor (PRLR) contributes to the progression of uterine carcinoma. However, the underlying mechanisms are not well understood, and current means of targeting the PRL/PRLR axis in uterine cancer are limited. Our integrated analyses using The Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) databases demonstrated that a short form of PRLR (PRLR_SF) is the isoform predominantly expressed in human uterine cancers; expression of this PRLR_SF was elevated in uterine cancers in comparison with cancer-free uterine tissues. We hypothesized that the overexpression of PRLR_SF in uterine cancer cells contributes, in part, to the oncogenic activity of the PRL/PRLR axis. Next, we employed G129R, an antagonist of human PRL, to block the PRL/PRLR axis in both PTENwt and PTENmut orthotopic mouse models of uterine cancer. In comparison with control groups, treatment with G129R as monotherapy or in combination with paclitaxel resulted in a significant reduction of growth and progression of orthotopic uterine tumors. Results from protein profiling of uterine cancer cells and in vivo tumors revealed a set of new downstream targets for G129R. Our results showed that G129R induced sub-G0 population arrest, decreased nascent protein synthesis, and initiated FOXO3a/EIF-4EBP1–mediated cell death in both PTENwt and PTENmut uterine cancer cells. Collectively, our results show a unique pattern of PRLR_SF expression predominantly in uterine cancer. Moreover, FOXO3a and EIF-4EBP1 are important mediators of cell death following G129R treatment in uterine cancer models.
