Breast Cancer Patient-derived Xenograft Tumors Research Articles

LncRNA BREA2 promotes metastasis by disrupting the WWP2-mediated ubiquitination of Notch1

Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.

Abstract PD8-02: Kinome profiling of ER+ breast cancer PDXs identifies PKMYT1 as a marker of hormone independent growth and poor outcome

Abstract Background: Endocrine therapy resistance is common and is a leading cause of breast cancer-related death. Thus, the search for therapeutic targets based on mechanistic insights into endocrine therapy resistance continues. Kinases are important drug targets and regulators in cells, many of which are involved in tumorigenesis and the development of treatment resistance. Mass spectrometry-based kinome analysis has been impeded by the low abundance of individual kinases. Here we utilized kinase inhibitor-conjugated beads to enrich and thereby sensitively profile the kinome of estrogen receptor-positive (ER+) breast cancer patient-derived xenograft (PDX) tumors under estradiol-deprivation treatment. Experimental design and methods: We harvested tumor samples from 20 breast PDX lines with various degrees of estradiol (E2) dependence in ovariectomized SCID-beige mice with and without E2 supplementation (n=3 per PDX line per arm). The kinases in the tumor lysates were enriched using kinase inhibitor pulldown (KIP) beads and the tightly bound kinases were quantified by mass spectrometry. To identify candidates, we selected for kinases that were highly E2-regulated in the E2-dependent PDX lines but constitutively expressed in E2-independent PDX lines. Survival analysis of candidate kinases in patients with ER+ breast cancer was performed using the METABRIC dataset. Results: Each PDX line had a unique yet reproducible kinome. To seek kinases with consistent relationships with estrogen dependence, we sought kinases that were statistically differentially expressed in tumors between E2 supplied and deprived conditions. Noticeably, membrane-associated tyrosine-and threonine-specific cdc2-inhibitory kinase (PKMYT1), a WEE family kinase known to have estrogen response elements (EREs) in its regulatory region, was significantly decreased after E2 deprivation in E2-dependent PDXs (log2 fold change = -7.86, p<0.001) but was constitutive in E2-independent PDXs. High PKMYT1 mRNA expression was associated with poor prognosis in the ER+ samples in METABRIC (hazard ratio=2.2, p<0.001). In contrast, the more studied member of the WEE family, WEE1, lacks an estrogen response element (ERE) and is not E2-regulated from the kinome profiling result. WEE1 mRNA expression level is not associated with the outcome of patients with ER+ breast cancer, suggesting that PKMYT1 has evolved a specific role in the cell cycle of ER+ tumors. Conclusion: Here, we analyzed the kinomes of 20 ER+ breast cancer PDX tumors with or without E2 by mass spectrometry. We discovered that PKMYT1 is a marker of hormone independent growth and poor outcome. Ongoing experiments that study the effects of PKMYT1 inhibition in both ER-dependent and independent circumstances will be presented. Citation Format: Anran Chen, Beom-Jun Kim, Doug W Chan, Purba Singh, Lacey E Dobrolecki, Jonathan T Lei, Shunqiang Li, Alana L Welm, Michael T Lewis, Matthew J Ellis. Kinome profiling of ER+ breast cancer PDXs identifies PKMYT1 as a marker of hormone independent growth and poor outcome [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-02.

Abstract 3687: Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells

Abstract ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic surface antigen that is expressed on breast cancer, but not on normal postpartum tissues. We found ROR1 was a receptor for Wnt5a, and high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). More recently, ROR1 could activate RhoGTPases and enhance tumor growth in breast cancer (Zhang S., et al., PNAS, 116(4), 2019). Such effects of Wnt5a on breast cancer patient-derived-xenograft (PDX) tumors could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. However, the molecular mechanism(s) for how ROR1 contributes such functions remained elusive. We performed mass spectrometry-based proteomic analysis on anti-ROR1 immune precipitates from breast cancer PDX tumors and detected DOCK1 in addition to ROR1, implying that ROR1 associates with DOCK1. DOCK1 (Dedicator of cytokinesis 1, also known as DOCK180) that expressed in breast cancer PDXs and cell lines, is a cytoplasmic protein, and a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1. Moreover, DOCK1 consists SH3 domain, which allows it to bind characteristic motifs (-P-X-X-P-), which often are found in the proline rich domains (PRD) of other proteins. Immunoblot analysis confirmed that ROR1 associated with DOCK1 in breast cancer PDX tumors or MCF7 cells transfected to express ROR1 (MCF7-ROR1) in response to Wnt5a, leading to enhanced activation of Rac1. To explore functional role, we performed a 2-week colony-formation assay using MCF7-ROR1 cells, and found that treatment of Wnt5a could enhance number of colonies of MCF7-ROR1 cells and that such colony formation capability could be inhibited by reducing expression of DOCK1 with specific siRNA, but not by control siRNA, suggesting a dependency on DOCK1 for Wnt5a-enhanced MCF7-ROR1 cells colony formation capability. Moreover, such effects of Wnt5a to enhance colony formation ability of MCF7-ROR1 cells could be inhibit by cirmtuzumab. We also introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1 proline-rich-domain (PRD) at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P⇒A mutants, ROR1P(808)A was unable to recruit DOCK1 to ROR1, or activate Rac1 in response to Wnt5a. Next we examined the tumor-growth potential of MCF7-Ctrl, MCF7-ROR1, or MCF7-P808A cells. The cells (5 × 104 cells/inoculate in RPMI 1640 together with an equal volume of Matrigel) were injected subcutaneously into the second right mammary pad of 4-to 6-week-old Rag2−/−γc−/- mice pretreated with estrogen pellets (17β-estradiol), which were placed subcutaneously in the intrascapular region one week before the cells inoculation. We found that MCF7-ROR1 cells (n=6) had significantly higher tumor growth relative to MCF7 cells (n=8), which had the same tumor growth as that of MCF7-P808A cells (n=6). Moreover, MCF7-ROR1P808A tumors had less activation of Rac1 compared to MCF7-ROR1 cells expressing wild-type ROR1. This study reveals that the recruitment of DOCK1 to ROR1 may be critical for the capacity of Wnt5a to enhance breast cancer tumor growth. Citation Format: Md Kamrul Hasan, George F. Widhopf II, Suping Zhang, Sharon M. Lam, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3687.

QPI Allows in vitro Drug Screening of Triple Negative Breast Cancer PDX Tumors and Fine Needle Biopsies

The development of resistance to initially successful cancer therapies is a major cause of the morbidity and mortality associated with cancer. Identifying evolving resistance at an early stage could inform clinical decision making to adapt therapies before resistant cancer cell phenotypes have become clonally dominant or metastasized. This goal of early detection has prompted heavy investments in liquid biopsy, organoid, and high-throughput screening methodologies. Recently, High-Speed Live-Cell Interferometry (HSLCI), a quantitative phase imaging (QPI) methodology, was shown to predict triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) sensitivity to carboplatin only 40 hours after tumor removal from a mouse. Before HSLCI can be tested in the clinic, it must be adapted for minimally invasive sample acquisition techniques, throughput must be increased to enhance drug screening capacity, and, like any screening method, protocols must be adjusted to reflect drug-specific effects. To overcome these barriers, the system’s hardware was redesigned to increase throughput six-fold and enable the simultaneous screening of multiple therapeutics, and experiments were expanded to include several new classes of drugs. Additionally, the updated system was then incorporated into fine needle biopsy-compatible protocols that were developed for HSLCI and yielded data concordant with recently-published in vivo screens.

Adipose-derived stem cells enhance human breast cancer growth and cancer stem cell-like properties through adipsin.

Tumor microenvironment plays a key role for tumor development and progression. Although adipose tissue is a predominant component of stroma in mammary tissues and secretes various cytokines, chemokines and growth factors, roles of adipocytes in breast cancers remain to be elucidated. In this study, we found that adipsin, an adipokine secreted from mammary adipose tissues, enhanced proliferation and cancer stem cell (CSC)-like properties of human breast cancer patient-derived xenograft (PDX) cells. Adipsin was predominantly expressed in both adipose tissues of the surgical specimens of breast cancer patients and adipose-derived stem cells (ADSCs) isolated from them, and its expression level was significantly higher in obese patients. ADSCs significantly enhanced the sphere-forming ability of breast cancer PDX cells derived from both estrogen receptor-positive and -negative breast cancer PDX cells. Suppression of adipsin-mediated signaling by a specific inhibitor or adipsin knockdown in ADSCs significantly decreased the sphere-forming ability and the expression of CSC markers in co-cultured breast cancer PDX cells. Growth of breast cancer PDX tumors was significantly enhanced by co-transplantation with ADSCs in vivo, and it was weakened when co-transplanted with the adipsin knocked-down ADSCs. These results suggest that adipsin is an important adipokine secreted from mammary adipose tissue that functions as a component of tumor microenvironment and a CSC niche in breast cancers.

Abstract 3492: Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on breast cancer, but not on virtually all normal adult tissues. Studies have found that high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival compared to that of patients with tumors that had low expression of ROR1 (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). Analysis of ROR1-immune precipitates of primary breast cancer patient-derived-xenograft (PDX) lysates via immunoblot analyses revealed that ROR1 was associated with DOCK1 (Dedicator of Cytokinesis 1, also known as DOCK180) in breast cancer PDX tumors. DOCK1 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 that is expressed in breast cancer PDX tumors or cell lines. DOCK1 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK1 in PDX cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a. Wnt5a could induce ROR1 to complex with DOCK1 and cause activation of Rac1; this effect could be inhibited by silencing DOCK1 in PDX cells. Furthermore, these effects of Wnt5a on PDX cells also could be blocked by treatment of the PDX cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. We corroborated these findings using the breast cancer cell-line MCF7, which also expresses DOCK1, but does not express ROR1. MCF7 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK1 interactions. We confirmed that DOCK1 complexes with ROR1 in response to Wnt5a in MCF7-ROR1 cells. We find that silencing DOCK1 specifically impaired the capacity of Wnt5a to enhance growth of MCF7-ROR1 cells in vitro. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK1 and activate Rac1 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P->A mutants, ROR1P(808)A had impaired capacity to recruit DOCK1 to ROR1 in response to Wnt5a. Moreover, unlike MCF7 cells transfected with wild-type ROR1 or ROR1 with P->A substitutions at positions 784, 826, or 841, MCF7 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MCF7 cells that do not express ROR1. This study reveals that the recruitment of DOCK1 may be critical for the capacity of Wnt5a to enhance breast cancer cell growth, which may contribute to the observed increased tendency for disease progression in breast cancer patients who have tumors that express high-levels of ROR1. Citation Format: Md Kamrul Hasan, Victoria Tripple, George F. Widhopf, Suping Zhang, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3492.

Abstract 92: CHIP/STUB1 ubiquitin ligase targets MZF1 and loss of its expression in breast cancer unleashes a MZF1-cathepsin pro-oncogenic program

Abstract Triple-negative and ErbB2+ breast cancer subtypes carry inherently poorer prognosis. The overall therapeutic response and survival are markedly lower in metastatic disease. This identification of novel pathways that collide with driver oncogenes to promote more aggressive disease and metastasis could help identify biomarkers for selection of patients for more intensive therapy and provide avenues of targeted therapy for new combinatorial therapies. CHIP/STUB1 is a U-box containing ubiquitin ligase that has been established as a negative co-chaperone for HSP90/HSC70. Recent studies have shown its expression to be reduced or lost in several cancers, including breast cancer. However, the relationship of loss of CHIP with breast cancer subtypes and mechanisms by which loss of CHIP promotes oncogenesis in breast cancer are incompletely understood. IHC analysis of an extensive breast cancer tissue collection revealed that loss of nuclear but not cytoplasmic CHIP predicted aggressive tumorigenesis and shorter survival, with loss of nuclear CHIP in two-thirds of TNBC/ErbB2+ and one-third of ER+ breast cancers. Reduced CHIP expression was confirmed in a panel of breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in TNBC and ErbB2+ breast cancer cell lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth in nude mice. These analyses established CHIP as a barrier to tumorigenic progression and loss of CHIP as a key event in aggressive tumorigenesis in breast cancer. To gain mechanistic insights into the linkage of the loss of nuclear CHIP expression with breast cancer aggressiveness, we carried out an unbiased screen for CHIP-regulated nuclear transcription factor-binding to their cognate DNA elements using an array methodology. This analysis identified many candidate transcription factors whose DNA-binding activity was up- or down-regulated upon re-expression of CHIP in CHIP-low ErbB2+ or TNBC cell line models. Western blotting of selected candidates confirmed the CHIP dependence of their expression levels. We focused our further efforts on the Myeloid Zinc Finger 1 (MZF1) protein as a CHIP target since it was recently identified as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion in a model of oncogenic mutant ErbB2 overexpression in MCF-7 cells. We show that CHIP negatively regulates the expression of CTSB/L in TNBC and other breast cancer cell lines. Chemical inhibition of CTSB abrogated breast cancer cell invasion and matrix degradation in vitro and retarded xenograft tumor growth. We suggest that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways that can provide new therapeutic opportunities in breast cancer. Citation Format: Bhopal C. Mohapatra, Haitao Luan, Timothy A. Bielecki, Insha Mushtaq, Sameer Mirza, Tameka A. Bailey, Robert Clubb, Wei An, Dena Ahmed, Rokaya El Ansari, Matthew D. Storck, Chittibabu Guda, Yuri Sheinin, Jane L. Meza, Srikumar Raja, Emad Rakha, Vimla Band, Hamid Band. CHIP/STUB1 ubiquitin ligase targets MZF1 and loss of its expression in breast cancer unleashes a MZF1-cathepsin pro-oncogenic program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 92.

Loss of the Nuclear Pool of Ubiquitin Ligase CHIP/STUB1 in Breast Cancer Unleashes the MZF1-Cathepsin Pro-oncogenic Program

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2+ and triple-negative breast cancers (TNBC) and in one third of ER+ breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in ErbB2+ lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2+ and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2+ breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression.Significance: These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524-35. ©2018 AACR.

Pathway-Enriched Gene Signature Associated with 53BP1 Response to PARP Inhibition in Triple-Negative Breast Cancer.

Effective treatment of patients with triple-negative (ER-negative, PR-negative, HER2-negative) breast cancer remains a challenge. Although PARP inhibitors are being evaluated in clinical trials, biomarkers are needed to identify patients who will most benefit from anti-PARP therapy. We determined the responses of three PARP inhibitors (veliparib, olaparib, and talazoparib) in a panel of eight triple-negative breast cancer cell lines. Therapeutic responses and cellular phenotypes were elucidated using high-content imaging and quantitative immunofluorescence to assess markers of DNA damage (53BP1) and apoptosis (cleaved PARP). We determined the pharmacodynamic changes as percentage of cells positive for 53BP1, mean number of 53BP1 foci per cell, and percentage of cells positive for cleaved PARP. Inspired by traditional dose-response measures of cell viability, an EC50 value was calculated for each cellular phenotype and each PARP inhibitor. The EC50 values for both 53BP1 metrics strongly correlated with IC50 values for each PARP inhibitor. Pathway enrichment analysis identified a set of DNA repair and cell cycle-associated genes that were associated with 53BP1 response following PARP inhibition. The overall accuracy of our 63 gene set in predicting response to olaparib in seven breast cancer patient-derived xenograft tumors was 86%. In triple-negative breast cancer patients who had not received anti-PARP therapy, the predicted response rate of our gene signature was 45%. These results indicate that 53BP1 is a biomarker of response to anti-PARP therapy in the laboratory, and our DNA damage response gene signature may be used to identify patients who are most likely to respond to PARP inhibition. Mol Cancer Ther; 16(12); 2892-901. ©2017 AACR.

Abstract B30: Ex vivo culture of breast cancer patient-derived xenografts in modified 3-D hyaluronan hydrogels

Abstract The high attrition rate of potential anti-cancer drugs entering clinical trials indicates the need for more predictive pre-clinical model systems. Standard compound screening is high-throughput and relatively inexpensive, but conducted with non-representative cells in a non-physiological environment. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment including relevant extracellular matrix (ECM) cues and cell-cell interactions. Patient-derived xenograft (PDX) culture preserves tumor heterogeneity while mitigating harsh selective pressures induced by traditional 2D culture methods. However, in vivo mouse models are slow and expensive, often with low “take” rates that limit the numbers of drugs and drug combinations that can be tested. Within the present study, we established the direct culture of breast cancer PDX cells in a hyaluronan-based hydrogel matrix that is amenable to automated dispensing. Washington University Human in Mouse (WHIM) breast cancer PDX tumors were dissociated and PDX cell were encapsulated in 3D hydrogels for in vitro culture. In vitro cultured PDX cells showed high viability over 7 days, as assessed by standard live/dead assay. Cell morphology and marker retention were also examined using immunofluorescence staining for markers including ER, EpCAM, and vimentin, and were found to be subtype-consistent. Ongoing studies seek to expand the PDX tumors in hydrogel matrices such that they may be useful for a variety of applications including drug screening, proteome and kinome activation analysis, and other applications that will enable the development of personalized therapies suited to individual breast cancer patients presenting with various disease subtypes. Citation Format: Lindsey K. Sablatura, MeghaShyam Kavuri, Priya Richards, Daniel A. Harrington, Matthew J. Ellis, Mary C. Farach-Carson. Ex vivo culture of breast cancer patient-derived xenografts in modified 3-D hyaluronan hydrogels. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B30.