Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poorer prognosis, higher tumour grade and greater tumour burden (1). Despite the recent introduction of specific immunotherapies and PARP inhibitors for treatment of certain subpopulations of TNBC patients, there remains a paucity of targeted treatments for TNBC and cytotoxic chemotherapy is still the cornerstone of treatment (2). Consequently, there is an urgent need to identify novel targeted and personalized treatment strategies for patients suffering from TNBC.
In order to identify protein kinases that may regulate progression of TNBC and represent potential therapeutic targets, we undertook mass spectrometry (MS)-based proteomic profiling across a panel of 24 TNBC cell lines to identify protein kinases with increased expression/activation in particular cell line subsets. In a recently published Oncogene paper (3), we report that this led to the identification of the multidomain pseudokinase Nuclear receptor binding protein 1 (NRBP1) on the basis that it exhibited marked variation in protein expression across the panel.
Over the last decade, evidence has emerged that NRBP1 plays context-specific roles in a variety of cancers, including colorectal, lung, prostate, bladder and breast cancers (4-9). In breast cancer, the role of NRBP1 remains controversial. One study reported that NRBP1 negatively regulates cell proliferation in two breast cancer cell lines (7). However, this is contradicted by data from comprehensive functional genomic screens across cancer cell lines identifying NRBP1 as a context-specific fitness gene, specifically in breast cancer (4).
In the Oncogene manuscript we present strong evidence from both in vitro and in vivo model systems as well as patient cohorts that NRBP1 plays an oncogenic role in TNBC. Interrogation of publicly-available data revealed that high NRBP1 expression positively correlates with poor distant disease-free and overall survival of TNBC patients, which indicates a potential role for NRBP1 in promoting TNBC progression and highlighted this protein for further functional and mechanistic characterization.
NRBP1’s oncogenic role was supported by our functional characterization of NRBP1 in cell culture models of TNBC using a variety of biological assays. Overexpression of NRBP1 enhanced cell migration and invasion, while knockdown of NRBP1 reduced cell proliferation, colony formation, migration and/or invasion. In addition, knockdown of NRBP1 in MDA-MB-231_HM cells markedly reduced xenograft growth and tumour metastasis to lung in BALB/c nude mice.
To address NRBP1’s signalling mechanism, we defined the interactome of NRBP1 in TNBC, identifying P-Rex1, Thioredoxin-like 1 (TXNL1) and Peroxiredoxin (PRD)1-3 as top-ranked candidates. Upon pathway analysis of NRBP1 interactors, reactive oxygen species (ROS)-related and cytoskeleton-regulated processes were identified as the top ten enriched functional categories. Since P-Rex1 is a known regulator of cytoskeletal organization and cell migration, positively regulates ROS production and promotes breast cancer development and metastasis (10-12), we selected this candidate for further characterization.
P-Rex1 is best-characterized as a guanine nucleotide exchange factor for the Rho family GTPase Rac1, although activity against Cdc42 has been reported (13-16). Both GTPases are key players in cell growth, migration, invasion and metastasis (14). Consequently, key questions were how does NRBP1 impact Rac1/Cdc42 activity, and the P-Rex1 dependency of NRBP1-mediated biological effects. To address the first question, pulldown activity assays were performed using lysates from TNBC cells, revealing that NRBP1 overexpression increased the activation levels of Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42. These data suggested a novel NRBP1/P-Rex1/Rac1/Cdc42 signalling axis in TNBC, that may involve a scaffolding function of NRBP1.
To further interrogate the requirement for the P-Rex1/Rac1/Cdc42 signalling axis in NRBP1-mediated biological effects, we used a variety of biological assays. These identified that NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, and active Rac1/Cdc42 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. These data indicated that the P-Rex1/Rac1/Cdc42 pathway is required for NRBP1-mediated oncogenic endpoints.
Since ROS-related processes represented major enriched functional categories for NRBP1 interactors, and P-Rex1 and Rac1/Cdc42 are known to promote ROS generation in a context-specific manner (17-19), we also characterized the role of ROS in NRBP1-regulated biological processes and found that ROS generation via the NRBP1/P-Rex1/Rac1/Cdc42 signalling axis was implicated in the oncogenic functions of NRBP1.
In conclusion, the findings in this paper define the oncogenic functions of NRBP1 in TNBC and a novel oncogenic signalling pathway that may be amenable to therapeutic intervention.
- Garrido-Castro AC, Lin NU, Polyak K. Insights into Molecular Classifications of Triple-Negative Breast Cancer: Improving Patient Selection for Treatment. Cancer Discov. 2019;9(2):176-98.
- Azim HA, Ghosn M, Oualla K, Kassem L. Personalized treatment in metastatic triple‐negative breast cancer: The outlook in 2020. The Breast Journal. 2020;26(1):69-80.
- Yang X, Cruz MI, Nguyen EV, Huang C, Schittenhelm RB, Luu J, et al. The pseudokinase NRBP1 activates Rac1/Cdc42 via P-Rex1 to drive oncogenic signalling in triple-negative breast cancer. Oncogene. 2023.
- Behan FM, Iorio F, Picco G, Gonçalves E, Beaver CM, Migliardi G, et al. Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature. 2019;568(7753):511-6.
- Liao Y, Yang Z, Huang J, Chen H, Xiang J, Li S, et al. Nuclear receptor binding protein 1 correlates with better prognosis and induces caspase-dependent intrinsic apoptosis through the JNK signalling pathway in colorectal cancer. Cell death & disease. 2018;9(4):436.
- Ruiz C, Oeggerli M, Germann M, Gluderer S, Stocker H, Andreozzi M, et al. High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth. The Prostate. 2012;72(15):1678-87.
- Wei H, Wang H, Ji Q, Sun J, Tao L, Zhou X. NRBP1 is downregulated in breast cancer and NRBP1 overexpression inhibits cancer cell proliferation through Wnt/β-catenin signaling pathway. OncoTargets and therapy. 2015;8:3721.
- Wilson CH, Crombie C, Van Der Weyden L, Poulogiannis G, Rust AG, Pardo M, et al. Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation. The EMBO journal. 2012;31(11):2486-97.
- Wu Q, Zhou X, Li P, Wang W, Wang J, Tan M, et al. High NRBP1 expression promotes proliferation and correlates with poor prognosis in bladder cancer. Journal of Cancer. 2019;10(18):4270.
- Lawson CD, Donald S, Anderson KE, Patton DT, Welch HC. P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses. J Immunol. 2011;186(3):1467-76.
- Srijakotre N, Liu HJ, Nobis M, Man J, Yip HYK, Papa A, et al. PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis. Proc Natl Acad Sci U S A. 2020;117(45):28056-67.
- Srijakotre N, Man J, Ooms LM, Lucato CM, Ellisdon AM, Mitchell CA. P-Rex1 and P-Rex2 RacGEFs and cancer. Biochem Soc Trans. 2017;45(4):963-77.
- Cash JN, Davis EM, Tesmer JJG. Structural and Biochemical Characterization of the Catalytic Core of the Metastatic Factor P-Rex1 and Its Regulation by PtdIns(3,4,5)P3. Structure. 2016;24(5):730-40.
- Crosas-Molist E, Samain R, Kohlhammer L, Orgaz JL, George SL, Maiques O, et al. Rho GTPase signaling in cancer progression and dissemination. Physiol Rev. 2022;102(1):455-510.
- Rosenfeldt H, Vazquez-Prado J, Gutkind JS. P-REX2, a novel PI-3-kinase sensitive Rac exchange factor. FEBS Lett. 2004;572(1-3):167-71.
- Welch HCE, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR, Erdjument-Bromage H, et al. P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-Regulated Guanine-Nucleotide Exchange Factor for Rac. Cell. 2002;108(6):809-21.
- Acevedo A, Gonzalez-Billault C. Crosstalk between Rac1-mediated actin regulation and ROS production. Free Radic Biol Med. 2018;116:101-13.
- Tackenberg H, Moller S, Filippi MD, Laskay T. The Small GTPase Cdc42 Is a Major Regulator of Neutrophil Effector Functions. Front Immunol. 2020;11:1197.
- Wang X, Ke Z, Chen G, Xu M, Bower KA, Frank JA, et al. Cdc42-dependent activation of NADPH oxidase is involved in ethanol-induced neuronal oxidative stress. PLoS One. 2012;7(5):e38075.
Please sign in or register for FREE
If you are a registered user on Nature Portfolio Cancer Community, please sign in