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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., (© 2022. The Author(s).)

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., (©2020 American Association for Cancer Research.)

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., (© 2019 Sharma et al.)

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 TGFβ1. 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 TGFβ 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 TGFβ 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 TGFβ 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 TGFβ/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 ., (©2018 American Association for Cancer Research.)

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 IL2Rγ null (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 mm 3 , 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.

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 ., (©2018 American Association for Cancer Research.)

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.

Purpose: 9-Nitrocamptothecin (9-NC) and 12-nitrocamptothecin (12-NC) are synthetic structural analogues of camptothecin (CPT) which have been prepared to explore the structure/activity relationship of this group of compounds against a wide variety of experimental tumors. As part of our investigation of the pharmacology and the mechanism of tumor inhibition of these compounds, we examined the effect of route of administration on the distribution of tritium-labeled 9-NC and 12-NC, an active and a poor chemotherapeutic agent, respectively., Methods: Quantitative whole-body autoradiography was used and our results were compared with previous results obtained with the parent compound CPT., Results: These studies revealed that, independent of the route of administration, both CPT derivatives were rapidly distributed to gall bladder, gastrointestinal tract and kidney. The excretion from these organs was indicated by the high levels of radioactivity in urine (urinary bladder) and feces (large intestines). The studies also indicated that the distributions of 9-NC and 12-NC were qualitatively similar, but quantitatively higher uptake of radioactivity was observed in animals treated with 12-NC than in those treated with 9-NC at 30 min following treatment. With the exception of the late sampling time (12 h after administration), the accumulation of radioactivity in the lungs (bronchioles) of animals that received an intravenous (i.v.) dose of 9-NC or 12-NC was higher than those treated with an intramuscular (i.m.) dose. However, the retention of drug-derived radioactivity in the tumors of mice treated with an i.m. dose of 9-NC was higher than that in the tumors of i.v.-treated animals and was also higher than that in tumors of animals treated with 12-NC., Conclusions: These results suggest that higher accumulation of 9-NC in tumor tissues than of 12-NC may contribute to the more potent chemotherapeutic activity of the former agent. Our results also suggest that i.m. injection is a more effective route of administration than i.v. administration.

Camptothecin (CPT) inhibits the growth of a wide variety of experimental tumors. As a part of our exploration of this drug for use as a cancer chemotherapeutic agent, we studied the effect of route of administration on the absorption, distribution and tumor uptake of [3H]-CPT. The rate of disappearance of [3H]-CPT-derived radioactivity from blood during the first 48 h was highest following oral than following intravenous (i.v.) administration. Thereafter blood levels were low irrespective of route of administration. Considerable [3H]-CPT-derived radioactivity was detected in urine and feces up to 48 h after dosing. Distribution studies were conducted using quantitative whole-body autoradiography (WBA). These studies revealed that independent of the route of administration, [3H]-CPT was rapidly excreted in the bile (gallbladder) followed by elimination into the small and large intestinal tract. Levels of CPT-derived radioactivity in the kidneys were minimal and mostly localized in the renal pelvis. Hepatic concentrations of CPT were low and were almost equal to those of the tumor. The lungs of animals treated i.v. showed higher uptake of radioactivity than those treated intramuscularly or orally. Tumor/blood ratios were slightly higher following oral administration than following administration by other routes. This study indicates that CPT is primarily eliminated via the bile. The gastrointestinal tract is the major site of accumulation and excretion of CPT.

Several metastatic models have been developed using clonal selection of human malignant cells metastasizing into a specific organ in NIH-I Swiss immunodeficient mice. The organs of choice were the central nervous system (CNS), targeted by metastases of malignant melanoma, and the liver, with metastases of colon adenocarcinoma. Additional models of adrenal metastases by malignant melanoma, and CNS involvement by implanted human lung squamous carcinoma or lymphoblastoid cells, are also available. Organ metastases, as well as the effects of treatment, were confirmed by autopsies and histological examination of the tissues or by a surgical inspection of the liver. The treatment end points were established as the increases in survival times of treated mice relative to placebo-treated controls. Camptothecins injected i.m. or delivered via gastrointestinal tract inhibit the growth of CNS metastases and increase the survival of treated animals. 9-Amino-20(S)-camptothecin was effective in the CNS model and in the model of liver metastases. The drug increased 3.3- and 5.7-fold the survival rates relative to untreated controls with metastases of colon adenocarcinoma to the liver, and all camptothecins were significantly more effective than 5-fluorouracil, currently a drug of choice in treatment of this disease. The xenograft models of metastases are available for studies of drug passage through the blood-brain barrier optimization of drug delivery to the liver, and for the development of new camptothecin-based treatment strategies.

Organ environment has been shown to modulate the efficacy of chemotherapy. A nude mouse model was used to study the influence of the in vivo environment on the sensitivity to chemotherapy of a human melanoma. A melanoma clone, SB1A, was implanted in two different sites (adrenals and subcutis) of nude mice which were subsequently treated with high-dose cyclophosphamide. After treatment, a 42% complete response rate was observed for the adrenal implants. No response was seen in the subcutis despite the same chemotherapy administration. This result is not attributable to differences in drug concentration in the target sites, because of the high-dose chemotherapy used. This model shows that the in vivo environment modifies the sensitivity to chemotherapy of a malignant melanoma clone. Further research is needed to clarify the role of host factors and of specific cell-stroma interactions that modify the biological behavior of malignant cells.

We have shown recently that the plant alkaloid camptothecin and its derivatives inhibited growth of human carcinoma and melanoma cells in vitro and induced regression of advanced human malignant melanoma tumors growing in immunodeficient (nude) mice. Here, we have extended these studies to show that the camptothecin derivative 9-nitro-20(S)- camptothecin (9NC) induces complete regression of advanced breast carcinoma tumors growing in nude mice. We also report that 9NC inhibits growth of nontumorigenic and tumorigenic breast cells in vitro. However, flow cytometry studies show that 9NC elicits differential effects on the cell cycle of nontumorigenic and tumorigenic cells. In general, 9NC-treated nontumorigenic cells accumulate slowly at the G2 phase of the cell cycle with no cell death. In contrast, 9NC-treated tumorigenic cells transverse rapidly from G1 to S phase followed by cell death. Removal of 9NC from the cell cultures resulted in most nontumorigenic cells dividing, whereas tumorigenic cells continued to die after removal of 9NC. Taken together, the findings indicate a different response of nontumorigenic and tumorigenic breast cells to 9NC.

We have recently shown that the plant alkaloid 20(S)-camptothecin and its derivatives 9-nitro-20(S)-camptothecin(9NC) and 9-amino-20(S)-camptothecin(9AC) inhibit the growth of a variety of human tumors xenografted in nude mice. In this report, we demonstrate that 9NC and 9AC effectively inhibit growth, and subsequently induce regression, of human ovarian tumors grown in nude mice. Tumor regression is accompanied by degenerative changes in the tumor cells as assessed by microscopic observations of histological sections prepared from the tumors. Parallel experiments in vitro show that 9NC inhibits in a similar manner the growth of human ovarian carcinoma cells, regardless of their ability to induce tumors when xenografted in nude mice, and induces similar morphological changes in both non-tumorigenic and tumorigenic cells, as assessed by microscopic observation. Flow cytometry studies show that 9NC-induced growth inhibition of the non-tumorigenic cells is associated with accumulation of these cells in G2. In contrast, 9NC-induced growth inhibition of the tumorigenic cells is associated with the generation of cells containing a reduced DNA content, that is, cells programmed to die. In conclusion, camptothecins appear to be cytostatic for non-tumorigenic, but cytotoxic for tumorigenic cells, an important finding from viewpoints of cell biology, pharmacology and cancer chemotherapy.

The camptothecin derivatives 9-nitro-camptothecin (9NC) and 9-amino-camptothecin (9AC) inhibit similarly growth of HL-60, KG-1, and U-937 cells in vitro, whereas growth of THP-1 cells is inhibited by 9AC, but not by 9NC. Growth inhibition is accompanied by enlargement of cells which contain one (HL-60, THP-1) or more (KG-1, U-937) nuclei. Flow cytometry studies showed that 9NC-treated HL-60 and U-937 cells accumulate in the S and G2 phases of the cell cycle; then they die by apoptosis, with the HL-60 cells being more sensitive than U-937 cells to 9NC. In contrast, 9NC-treated KG-1 and THP-1 cells accumulate in S and G2 phases, but resist death by apoptosis. Of the cell lines tested, only U-937 cells xenografted in nude mice generated subcutaneous myeloid tumors, which exhibited a delayed growth in the presence of 9NC. Further, 9NC-treated advanced U-937 tumors regressed temporarily, indicating that U-937 cells consist of 9NC-sensitive and 9NC-resistant populations.

We have shown recently that the plant alkaloid camptothecin and some of its derivatives inhibit growth of human breast carcinoma cells in vitro and induce complete regression of human breast tumors grown in nude mice. Because of the use of camptothecin derivatives in several clinical studies with patients bearing various types of cancer, in this report, we have investigated and described parameters and conditions that can modulate the anticancer effectiveness and cytotoxicity of these drugs when administered as suspensions. It is demonstrated that the antitumor effectiveness and drug-induced toxicity depend on the camptothecin derivative, the drug dose administered, the route of administration, and the scheduling of drug administration. 9-aminocamptothecin administered i.m. generates the best results, but for all practical purposes, 9-nitrocamptothecin administered orally appeared to be the camptothecin derivative and route of administration of choice. Further, we report the partial response of one breast tumor to camptothecin and propose that this tumor may require chemotherapy with a camptothecin derivative followed by an anticancer drug with a different mechanism of cell-killing activity.

The plant alkaloid camptothecin and three camptothecin derivatives were used to study responses of human malignant melanoma (BRO) cells xenografted in immunodeficient (nude) mice. Camptothecin and its derivatives 9-nitro-20(S)-camptothecin and 9-amino-20(S)-camptothecin inhibited growth of tumors and caused regression in all tumor-bearing mice. Tumor regression was accompanied by degenerative changes in the tumor cells, as assessed by microscopic observations of histological sections prepared from the tumors. No toxic effects were observed in the drug-treated mice, with or without xenografts. In parallel experiments, camptothecin, 9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin inhibited proliferation of BRO cells in vitro and resulted in dramatic morphological cellular changes comparable to those observed in the sections of solid tumors. The derivative 12-nitro-20(S)-camptothecin had no effect on BRO tumors or cell cultures. The difference between 9-nitro-20(S)-camptothecin and 12-nitro-20(S)-camptothecin is the position at which the NO2 group is attached to the camptothecin molecule. In contrast to BRO melanoma cells, none of the camptothecin derivatives had any effect on cultured human melanocytes, the normal counterparts of melanoma cells. Taken together, the findings indicate that camptothecin and derivatives exert different effects on the growth and morphology of normal and malignant (BRO) melanocytes.

20-(S)-Camptothecin (CAM), a plant alkaloid, was tested against 13 human cancer xenograft lines carried by immunodeficient (nude) mice. The drug, formulated in 20% intralipid and given i.m., was more effective than any other clinically available drug tested. It was found that: (a) CAM, at nontoxic doses, suppressed growth and induced regression of cancer of the colon (3 lines), lung (4 lines), breast (2 lines), stomach (1 line), ovary (1 line), and malignant melanoma (2 lines); (b) the drug was equally effective administered i.m. or p.o. Both routes are significantly better than i.v. administration; (c) CAM is substantially more effective and less toxic than its sodium salt, which was unsuccessfully tested in cancer patients. CAM should be further tested against responsive cancers as a drug which is easy to isolate and formulate for large-scale studies.

The purpose of this study was to examine whether organ specific metastasis can be produced by different subpopulations of cells isolated from a single human melanoma. Three clones, SB1, SB2, SB3, and a variant line SB1 Asc (obtained from ascites fluid after i.v. inoculation of SB1 cells in a nude mouse) - all isolated from a primary human cutaneous melanoma - were inoculated into the left ventricle (i.c.) or intravenously (i.v.) into nude mice. Three to 16 weeks later, the animals were killed and autopsied. The clones produced different patterns of metastasis. Three to 4 weeks after i.c. inoculation of 2 x 10(6) cells, cells of clone SB1 produced numerous rapidly progressing metastases in the brain, whereas cells of SB1 Asc produced large metastases in the adrenals and sometimes in the ovaries. Cells of clones SB2 and SB3 produced a few small metastases without any characteristics pattern. The initial arrest of tumor cells in various organs did not predict the outcome of metastasis. This conclusion is based on studies of distribution and fate of tumor cells labeled with 111Indium Oxine. Therefore, organ specific metastasis produced by human melanoma cells inoculated i.c. into nude mice is likely to depend on the interaction of unique metastatic cells with organ environment.