Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models.

BACKGROUND: Breast cancer has been considered not highly immunogenic, and few patients benefit from current immunotherapies. However, new strategies are aimed at changing this paradigm. In the present study, we examined the in vivo activity of a humanized anti-programmed cell death protein 1 (anti-PD-1) antibody against triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tumor models. METHODS: To circumvent some of the limitations posed by the lack of appropriate animal models in preclinical studies of immunotherapies, partially human leukocyte antigen-matched TNBC PDX tumor lines from our collection, as well as human melanoma cell lines, were engrafted in humanized nonobese diabetic/severe combined immunodeficiency IL2Rγnull (hNSG) mice obtained by intravenous injection of CD34+ hematopoietic stem cells into nonlethally irradiated 3-4-week-old mice. After both PDXs and melanoma cell xenografts reached ~ 150-200 mm3, animals were treated with humanized anti-PD-1 antibody or anti-CTLA-4 and evaluated for tumor growth, survival, and potential mechanism of action. RESULTS: Human CD45+, CD20+, CD3+, CD8+, CD56+, CD68+, and CD33+ cells were readily identified in blood, spleen, and bone marrow collected from hNSG, as well as human cytokines in blood and engrafted tumors. Engraftment of TNBC PDXs in hNSG was high (~ 85%), although they grew at a slightly slower pace and conserved their ability to generate lung metastasis. Human CD45+ cells were detectable in hNSG-harbored PDXs, and consistent with clinical observations, anti-PD-1 antibody therapy resulted in both a significant reduction in tumor growth and increased survival in some of the hNSG PDX tumor lines, whereas no such effects were observed in the corresponding non-hNSG models. CONCLUSIONS: This study provides evidence associated with anti-PD-1 immunotherapy against TNBC tumors supporting the use of TNBC PDXs in humanized mice as a model to overcome some of the technical difficulties associated with the preclinical investigation of immune-based therapies.

Tocilizumab overcomes chemotherapy resistance in mesenchymal stem-like breast cancer by negating autocrine IL-1A induction of IL-6.

Triple-negative breast cancer (TNBC) patients with mesenchymal stem-like (MSL) subtype have responded poorly to chemotherapy whereas patients with basal-like 1 (BL1) subtype achieved the best clinical response. In order to gain insight into pathways that may contribute to the divergent sensitivity to chemotherapy, we compared the inflammatory profile of the two TNBC subtypes treated with docetaxel. Cellular signaling analysis determined that docetaxel activated MAPK pathway in MSL TNBCs but not BL1 TNBCs. The subsequent MAPK pathway activation in MSL TNBCs led to an IL-1A mediated cascade of autocrine inflammatory mediators including IL-6. Utilizing the humanized IL-6R antibody, tocilizumab, our in vitro and in vivo data show that MSL TNBCs treated with tocilizumab together with chemotherapy results in delayed tumor progression compared to MSL TNBCs treated with docetaxel alone. Our study highlights a molecular subset of TNBC that may be responsive to tocilizumab therapy for potential translational impact.

SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.

A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

Antihistamine Drug Ebastine Inhibits Cancer Growth by Targeting Polycomb Group Protein EZH2.

Enhancer of zester homolog 2 (EZH2), a histone lysine methyltransferase and the catalytic component of polycomb repressive complex 2, has been extensively investigated as a chromatin regulator and a transcriptional suppressor by methylating H3 at lysine 27 (H3K27). EZH2 is upregulated or mutated in most cancers, and its expression levels are negatively associated with clinical outcomes. However, the current developed small-molecule inhibitors targeting EZH2 enzymatic activities could not inhibit the growth and progression of solid tumors. Here, we discovered an antihistamine drug, ebastine, as a novel EZH2 inhibitor by targeting EZH2 transcription and subsequently downregulating EZH2 protein level and H3K27 trimethylation in multiple cancer cell lines at concentrations below 10 µmol/L. The inhibition of EZH2 by ebastine further impaired the progression, migration, and invasiveness of these cancer cells. Overexpression of Ezh2 wild-type and its mutant, H689A (lacking methyltransferase activity), rescued the neoplastic properties of these cancer cells after ebastine treatment, suggesting that EZH2 targeted by ebastine is independent of its enzymatic function. Next-generation RNA-sequencing analysis also revealed that C4-2 cells treated with 8 µmol/L ebastine showed a gene profiling pattern similar to EZH2-knockdown C4-2 cells, which was distinctively different from cells treated with GSK126, an EZH2 enzyme inhibitor. In addition, ebastine treatment effectively reduced tumor growth and progression, and enhanced progression-free survival in triple-negative breast cancer and drug-resistant castration-resistant prostate cancer patient-derived xenograft mice. Our data demonstrated that ebastine is a novel, safe, and potent anticancer agent for patients with advanced cancer by targeting the oncoprotein EZH2.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

Activating Transcription Factor 4 Modulates TGFß-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFß1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFß and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFß and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFß was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFß/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697-709. ©2018 AACR.

Preclinical evaluation of the anticancer activity and toxicity of 9-nitro-20(S)-camptothecin (Rubitecan).

The purpose of this study is to establish the maximum tolerated dose of rubitecan in mice, dogs and men and to establish the anticancer activity of such dose against human tumors xenografted in nude mice. Nude mice received increasing doses of Rubitecan by intrastomach injection until the maximum tolerated dose (MTD) had been established for both the single dose and the multiple doses at the schedule of 5 days on, 2 days off. Extrapolating from the mouse data, MTD was determined for oral administration in dogs and man. Levels of the drug in plasma were determined by high pressure liquid chromatography (HPLC). Using maximum tolerated multiple doses, the sensitivity of human cancer xenografts in nude mice to Rubitecan was determined. MTD of Rubitecan in mice for multiple doses intrastomach at the schedule of 5+,2- was 1 mg/kg/day. MTD in dogs was also 1 mg/kg/day, administered orally but at the schedule of 4+,3-. In man, it was 1 mg/m2/day at the schedule of 5+,2-. Treatment of human cancer xenografts in nude mice with MTD of Rubitecan resulted in 100% growth inhibition of 30/30 tumors tested and in 24/30 in their total disappearance. These 30 tumors comprised all the most common human cancers: lung, colorectal, breast, pancreatic, ovarian, prostate, stomach, melanoma and a leukemia. From the data collected, it appears that rubitecan is a very promising anticancer drug with high potency against a wide spectrum of human cancers. These cancers growing as xenografts in nude mice are always growth inhibited (30/30) and frequently (24/30) totally destroyed by the administration of non-toxic doses of Rubitecan.