Abstract PD6-01: Cooperation of clinically-relevant genomic alterations transforms mammary progenitor cells to metaplastic breast cancer
Authors: Hicks M , Owens P , Moore P , Giltnane J , Estrada M , Sanders M , Cook R , Arteaga C , Balko J 
Published: May 2016

Background: We have previously identified the MAPK phosphatase dual specificity phosphatase 4 (Dusp4) as frequently down-regulated in triple-negative and basal-like breast cancer (TNBC and BLBC, respectively). DUSP4 is deregulated in BLBCs by heterozygous and homozygous loss, as well as by promoter CpG methylation. In TNBC and BLBC, DUSP4 deregulation frequently co-occurs with MYC amplification and mutations in TP53. In addition, loss of DUSP4 promotes activation of ETS-1 and cJUN, transcription factors downstream of Ras/MAPK and JNK, while imparting resistance to chemotherapy and promoting tumor-initiating cells, suggesting DUSP4 is a tumor suppressor. Thus, we hypothesized that loss of Dusp4 in the mammary epithelium, together with MYC amplification and mutations in Trp53, would promote tumorigenesis.

Methods: We generated transgenic mice with a conditional floxed Dusp4 allele (Dusp4FLOX) and isolated mammary epithelial progenitor cells. The resulting cells were transduced with adenovirus to deliver either Cre recombinase to excise the Dusp4 locus (Dusp4NULL) or GFP control. CRISPR-mediated mutagenesis (or non-targeting CRISPR control) was used to disrupt exon 7 or 8 of Trp53 causing relevant p53 mutations and MYC was overexpressed (or LACZ control). Isogenic cell lines were assessed for tumorigenicity, ploidy, sensitivity to chemotherapy, proliferation, and cell signaling.

Results: The isolated parental (Dusp4FLOX) mammary cell line maintained proliferation through &gt;50 passages without senescing and was highly cytokeratin (Ck) 5/14+ and moderately Ck 8/18+, suggestive of a bipotent progenitor-like lineage. As expected, p53 disruption, regardless of Dusp4 status, resulted in tetraploidy and loss of p21 expression. With concurrent Dusp4, p53 and MYC alterations, cells became increasingly resistant to chemotherapy and targeted agents, as measured with proliferation assays. In order to determine whether cells formed tumors in vivo, we implanted isogenic cell lines subcutaneously into nude mice. We found that only Dusp4NULL cells, together with p53 disruption and MYC overexpression (DPM) generated aggressive tumors (forming and ulcerating within 1-2 weeks), while the Dusp4FLOX counterparts (p53 disruption and MYC overexpression; PM) did not. Syngeneic transplantation revealed that DPM cells could also form tumors in an immune competent setting. Cells with Dusp4 loss and p53 mutation were also able to develop tumors without MYC amplification, but at a much slower rate. Tumors derived from the DPM cells were primarily metaplastic or basal-like in nature, including intra-tumor heterogeneity (pockets of Ck5 and pockets of Ck8, with regions of Ck8+ keratin pearls).

Conclusions: We have developed a genetically relevant mouse model to study BLBC in immune competent mice for unique opportunities to understand the pathogenesis and therapy response in BLBC, including immunotherapy. These results support the hypothesis that DUSP4 loss promotes tumorigenesis in BLBC. An understanding of the mechanism whereby Dusp4 loss contributes to this process in vivo should provide a rational for novel therapeutic strategies to treat BLBCs with DUSP4 loss.

Citation Format: Hicks M, Owens P, Moore P, Giltnane J, Estrada M, Sanders M, Cook R, Arteaga C, Balko J. Cooperation of clinically-relevant genomic alterations transforms mammary progenitor cells to metaplastic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD6-01.