Abstract 4086: HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative breast cancer

Abstract

Abstract The role of HMGXB3 (HMG Box Domain Containing 3), member of the non-canonical high mobility group (HMG) genes, in cancer remains largely unknown. A preliminary “mutual exclusivity” analysis of the Breast Invasive TCGA database showed a significant association between HMGXB3 and TGFβ (Transforming Growth Factor β). TGFβ plays a significant role in metastasis and increases stem-like properties of breast cancer cells. This work aimed at elucidating whether there is a relationship between HMGXB3 and TGFβ, and their role on breast cancer stem cells (BCSC) and metastasis in triple negative breast cancer (TNBC). Changes in HMGXB3 protein levels (Abgent) were assessed by Western blot in MCF10A cells treated with human recombinant (hr) TGFβ1 (R&D) with/without the TGFβR1 kinase inhibitor, LY2157299. Knockdown was performed in MCF10A and two TNBC cell lines, SUM159 and MDA-MB-231. Cells transfected with two different siRNAs against HMGXB3, siRNA#1 and siRNA#2, and one negative control (siRNA-SCR) were assayed for proliferation as well as primary and secondary mammosphere (MS) formation for 48h with hrTGFβ1 (10ng/ml). Cell migration was determined in transfected TNBC cell lines treated with 10ng/ml hrTGFβ1 for 72h. For in vivo experiments, MDA-MB-231, stably transfected with a luciferase/GFP reporter plasmid, were injected in the right mammary fat pad of female SCID Beige mice (n = 10). Tumor-bearing mice were treated with DOPC (dioleoyl phosphatidylcholine) liposomes loaded with 5μg of either siRNA-SCR or HMGXB3-siRNA#2 with/without docetaxel (20mg/kg) for 21 days. Tumors were digested and cells isolated. Single cells were assayed for aldefluor (ALDF+) by flow cytometry and MS formation. Lung metastases were determined in lungs by ex vivo imaging of luciferase positive cells. Our results show a concentration- and time-dependent induction of HMGXB3 protein levels under treatment with hrTGFβ1 in MCF10A cells. Such an enhancement was efficiently abrogated by the addition of LY2157299. Knockdown of HMGXB3 in presence of hrTGFβ1 significantly reduced cell proliferation, migration and MS formation in the TNBC cell lines tested herein. In vivo experiments demonstrated that silencing of HMGXB3 correlates with reduced tumor growth, ALDF+ BCSC subpopulation, and formation of secondary MS compared to siRNA-SCR. Finally, silencing HMGXB3 significantly abrogated progression of metastasis from primary tumor to lungs in contrast to siRNA-SCR with/without chemotherapy. In conclusion, we are the first to identify HMGXB3 as a TGFβ-induced cancer gene that impacts tumor cell proliferation, migration, BCSC self-renewal and lung metastases in TNBC. Our preliminary results suggest HMGXB3 as a novel target for prevention of metastasis. Future experiments will elucidate the role and mechanism of HMGXB3 in cancer. This work will give rise to novel therapeutic targets for the treatment of breast cancer. Citation Format: Sergio Granados-Principal, Yi Liu, Cristian Rodriguez-Aguayo, Anil K. Sood, Jenny C. Chang. HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2015-4086