ABSTRACT
PURPOSE: Limited knowledge exists on the detection of breast cancer stem cell (BCSC)-related mutations in circulating free DNA (cfDNA) from patients with advanced cancers. Identification of new cancer biomarkers may allow for earlier detection of disease progression and treatment strategy modifications. METHODS: We conducted a prospective study to determine the feasibility and prognostic utility of droplet digital polymerase chain reaction (ddPCR)-based BCSC gene mutation analysis of cfDNA in patients with breast cancer. RESULTS: Detection of quantitative BCSC gene mutation in cfDNA by ddPCR mirrors disease progression and thus may represent a valuable and cost-effective measure of tumor burden. We have previously shown that hematological and neurological expressed 1-like (HN1L), ribosomal protein L39 (RPL39), and myeloid leukemia factor 2 (MLF2) are novel targets for BCSC self-renewal, and targeting these genetic alterations could be useful for personalized genomic-based therapy. CONCLUSION: BCSC mutation detection in cfDNA may have important implications for diagnosis, prognosis, and serial monitoring.
Subject(s)
Biomarkers, Tumor , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Transformation, Neoplastic/genetics , Circulating Tumor DNA , Mutation , Neoplastic Stem Cells/metabolism , Adult , Aged , Aged, 80 and over , Breast Neoplasms/blood , Breast Neoplasms/mortality , DNA Mutational Analysis , Disease Progression , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Middle Aged , Neoplasm Grading , Neoplasm Metastasis , Neoplasm Staging , PrognosisABSTRACT
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.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Triple Negative Breast Neoplasms/therapy , Xenograft Model Antitumor Assays/methods , Animals , Cytokines/blood , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Female , Humans , Immunotherapy/methods , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Programmed Cell Death 1 Receptor/immunology , Triple Negative Breast Neoplasms/blood , Triple Negative Breast Neoplasms/immunology , Tumor Burden/drug effects , Tumor Burden/immunologyABSTRACT
BACKGROUND: Breast cancer mortality is most common in cancer in women, and there are no ex vivo models that can capture the primary growth of tumor with fidelity to the in vivo tumor growth. In this study, we grew human breast cancer cell lines in an acellular lung matrix of the ex vivo four-dimensional lung model to determine if they form primary tumor and the extent to which they mimic the histology and characteristics of the human tumors. MATERIALS AND METHODS: Rat lungs were harvested, decellularized, and placed in a bioreactor. To study the primary tumor growth, we seeded the lung via the trachea with human breast cancer cells SUM159, MCF7, or MDMB231 and perfused the pulmonary artery with oxygenated media. Lobectomies were performed and processed for hematoxylin and eosin, Ki-67, caspase-3, estrogen receptor, and progesterone receptor antibodies. RESULTS: All three cell lines grew in the ex vivo four-dimensional model and formed perfusable tumor nodules with similar histology and morphology as the primary tumors. SUM159 and MDAMB231 showed higher proliferation and apoptotic indices than MCF7. In addition, MCF7 retained its estrogen receptor and progesterone receptor positivity, whereas SUM159 and MDAMB 231 did not have any staining. CONCLUSIONS: Overall, our study showed that human breast cancer cells can be grown on the ex vivo four-dimensional lung model, which then form primary tumor nodules that mimic the morphology and histology of the original tumor.
Subject(s)
Breast Neoplasms/pathology , Lung/pathology , Animals , Apoptosis , Biomarkers, Tumor/metabolism , Bioreactors , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Female , Humans , In Vitro Techniques , Male , Neoplasm Invasiveness , Rats , Rats, Sprague-DawleyABSTRACT
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
Subject(s)
Breast Neoplasms/genetics , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Mutation/genetics , Paracrine Communication , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction , Amphiregulin/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatography, Liquid , Class I Phosphatidylinositol 3-Kinases , Disease-Free Survival , Down-Regulation/drug effects , Epidermal Growth Factor/pharmacology , ErbB Receptors/antagonists & inhibitors , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Humans , Mice, Nude , Neoplasm Proteins/metabolism , Paracrine Communication/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proteomics , Signal Transduction/drug effects , Tandem Mass Spectrometry , Up-Regulation/drug effectsABSTRACT
We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.
Subject(s)
Breast Neoplasms/metabolism , Lung Neoplasms/genetics , Neoplastic Stem Cells/cytology , Nitric Oxide Synthase/metabolism , Nuclear Proteins/metabolism , Ribosomal Proteins/metabolism , Animals , Breast Neoplasms/prevention & control , Cell Line, Tumor , Cell Movement , Female , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Hypoxia , Lung Neoplasms/metabolism , Mice , Mice, SCID , Mutation , Neoplasm Metastasis , Neoplasm Transplantation , Nitric Oxide/chemistry , Nitric Oxide Synthase/antagonists & inhibitors , RNA, Small Interfering/metabolism , Sequence Analysis, RNA , Signal Transduction , Time FactorsABSTRACT
INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor ß was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Triple Negative Breast Neoplasms/metabolism , Activating Transcription Factor 3/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Endoplasmic Reticulum Stress , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/secondary , Mice , Molecular Targeted Therapy , Neoplasm Invasiveness , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Prognosis , Transforming Growth Factor beta/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/mortality , Triple Negative Breast Neoplasms/pathology , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44(+) /CD24(-/low) cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44(+) /CD24(-/low) CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44(+) /CD24(-/low) CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus-activated kinase 2 (Jak2)-signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1.
Subject(s)
Chloroquine/pharmacology , DNA (Cytosine-5-)-Methyltransferases/metabolism , Janus Kinase 2/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Animals , Autophagy/drug effects , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , Female , Humans , Mice , Mice, Nude , Neoplastic Stem Cells/pathology , Signal Transduction/drug effects , Triple Negative Breast Neoplasms/metabolismABSTRACT
The estrogen receptor and human epidermal growth factor receptor (HER) signaling pathways are the dominant drivers of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, targeting these pathways provides the most effective therapies in appropriately selected patients. However, de novo and acquired resistance remain major obstacles to successful treatment. By increasing the understanding of the molecular mechanisms of combined HER2-targeted therapies, we aim to be better able to select patients who would respond to these treatments and understand some of the mechanisms of resistance to HER2-targeted treatments. Recent studies have demonstrated an increased effectiveness of dual targeted HER2 therapies against HER2-amplified breast cancer as compared with single blockade. These studies have resulted in the recent US Food and Drug Administration approval of the combination of taxane chemotherapy with pertuzumab and trastuzumab in the first-line metastatic setting as well as an accelerated approval in the neoadjuvant setting. Another mechanism for overcoming resistance to HER2 targeted therapies is the antibody-drug conjugate trastuzumab-emtansine, which targets the HER2 receptor conjugated to the potent antimicrotubule agent mertansine, allowing for intracellular release of the cytotoxic drug. Studies evaluating the efficacy of dual blockade with antibody-drug conjugate are currently ongoing. This article reviews recent data on different combinations of anti-HER2 treatments as well as ongoing and future research in this area.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/drug therapy , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/metabolism , Female , Gefitinib , Humans , Lapatinib , Molecular Targeted Therapy , Quinazolines/administration & dosage , Randomized Controlled Trials as Topic , Trastuzumab , Xenograft Model Antitumor AssaysABSTRACT
Sustained and complete inhibition of HER3 and its output to PI3K/Akt are required for the optimal antitumor effect of therapeutic inhibitors of the HER2 oncogene. Here, we show that, after inhibition of the HER2 tyrosine kinase with lapatinib, there is PI3K/Akt and FoxO3a-dependent up-regulation of HER3 mRNA and protein. Up-regulated HER3 was then phosphorylated by residual HER2 activity, thus partially maintaining P-Akt and limiting the antitumor action of lapatinib. Inhibition of HER3 with siRNA or a neutralizing HER3 antibody sensitized HER2+ breast cancer cells and xenografts to lapatinib both in vitro and in vivo. Combined blockade of HER2 and HER3 inhibited pharmacodynamic biomarkers of PI3K/Akt activity more effectively than each inhibitor alone. These results suggest that because of HER3-mediated compensation, current clinical inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway completely. They also suggest that therapeutic inhibitors of HER3 should be used in combination with HER2 inhibitors and PI3K pathway inhibitors in patients with HER2- and PI3K-dependent cancers.
Subject(s)
Breast Neoplasms/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/biosynthesis , Transcription, Genetic/drug effects , Up-Regulation/drug effects , Animals , Breast Neoplasms/drug therapy , Female , Humans , Lapatinib , Mice , Mice, Nude , Neoplasm Transplantation , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/antagonists & inhibitors , Transplantation, HeterologousABSTRACT
Breast cancer relapse, in a large number of patients, after initial response to standard of care therapy warrants development of novel therapies against recurrent and metastatic cancer. Cancer stem cells (CSCs), present in breast tumors while being intrinsically resistant to conventional therapy, have the ability to self renew and cause tumor recurrence. The residual tumors after therapy, with dramatic enrichment of the CSCs, have all the hallmarks of epithelial- mesenchymal transition (EMT). This review will focus on the link between EMT, CSCs and treatment resistance, since a better understanding of these interactions will allow us to effectively target the residual population after therapy.
Subject(s)
Breast Neoplasms/pathology , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Neoplastic Stem Cells/pathology , Animals , Breast Neoplasms/drug therapy , Cell Transformation, Neoplastic/pathology , Female , Humans , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/physiologyABSTRACT
Obesity is thought to contribute to worse disease outcome in breast cancer as a result of increased levels of adipocyte-secreted endocrine factors, insulin, and insulin-like growth factors (IGFs) that accelerate tumor cell proliferation and impair treatment response. We examined the effects of patient obesity on primary breast tumor gene expression, by profiling transcription of a set of 103 tumors for which the patients' body mass index (BMI) was ascertained. Sample profiles were stratified according to patients' obesity phenotype defined as normal (BMI < 25), overweight (BMI 25-29.9), or obese (BMI ≥ 30). Widespread gene expression alterations were evident in breast tumors from obese patients as compared to other tumors, allowing us to define an obesity-associated cancer transcriptional signature of 662 genes. In multiple public expression data sets of breast cancers (representing > 1,500 patients), manifestation of the obesity signature patterns correlated with manifestation of a gene signature for IGF signaling and (to a lesser extent) with lower levels of estrogen receptor. In one patient cohort, manifestation of the obesity signature correlated with shorter time to metastases. A number of small molecules either induced or suppressed the obesity-associated transcriptional program in vitro; estrogens alpha-estradiol, levonorgestrel, and hexestrol induced the program, while several anti-parkinsonian agents targeting neurotransmitter receptor pathways repressed the program. Obesity in breast cancer patients appears to impact the gene expression patterns of the tumor (perhaps as a result of altered body chemistry). These results warrant further investigation of obesity-associated modifiers of breast cancer risk and disease outcome.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/metabolism , Gene Expression Profiling , Obesity/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/complications , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Insulin-Like Growth Factor I/physiology , Kaplan-Meier Estimate , Obesity/complications , Obesity/genetics , Oligonucleotide Array Sequence Analysis , Proportional Hazards Models , Prospective Studies , Receptors, Estrogen/metabolism , Reference Values , Signal Transduction , Statistics, Nonparametric , Transcription, GeneticABSTRACT
The tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten(+/-) heterozygotes in either lesion development or progression. Lesions in both Pten(+/-); Tsc2(+/-), and Pten(+/-) mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten(+/-) mice. Furthermore, prostate lesion growth in Pten(+/-) mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment.
Subject(s)
Adenocarcinoma/pathology , PTEN Phosphohydrolase/physiology , Prostatic Neoplasms/pathology , Protein Kinases/metabolism , Tumor Suppressor Proteins/physiology , Adenocarcinoma/metabolism , Animals , Blotting, Western , Disease Progression , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Humans , Incidence , Loss of Heterozygosity , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsatellite Repeats , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/metabolism , Protein Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 2 ProteinABSTRACT
Dietary contribution to breast cancer risk, recurrence, and progression remains incompletely understood. Increased consumption of soy and soy isoflavones is associated with reduced mammary cancer susceptibility in women and in rodent models of carcinogenesis. In rats treated with N-methyl-N-nitrosourea, dietary intake of soy protein isolate (SPI) reduced mammary tumor occurrence but increased incidence of more invasive tumors in tumored rats, relative to the control diet casein. Here we evaluated whether mammary tumor progression in tumor-bearing rats lifetime exposed to SPI is associated with deregulated progesterone receptor (PR) isoform expression. In histologically normal mammary glands of rats with invasive ductal carcinoma lesions, PR-A protein levels were higher for SPI- than casein-fed rats, whereas PR-B was undetectable for both groups. Increased mammary PR-A expression was associated with higher transforming growth factor-beta1, stanniocalcin-1, and CD44 transcript levels; lower E-cadherin and estrogen receptor-alpha expression; and reduced apoptotic status in ductal epithelium. Serum progesterone (ng/ml) (CAS: 25.94 +/- 3.81; SPI: 13.19 +/- 2.32) and estradiol (pg/ml) (CAS: 27.9 +/- 4.49; SPI: 68.48 +/- 23.87) levels differed with diet. However, sera from rats of both diet groups displayed comparable mammosphere-forming efficiency in human MCF-7 cells. Thus, soy-rich diets may influence the development of more aggressive tumors by enhancing PR-A-dependent signaling in premalignant breast tissues.
Subject(s)
Genistein/administration & dosage , Isoflavones/administration & dosage , Mammary Neoplasms, Experimental/etiology , Receptors, Progesterone/physiology , Animals , Carcinoma, Ductal, Breast/etiology , Carcinoma, Intraductal, Noninfiltrating/etiology , Cell Line, Tumor , Disease Progression , Female , Humans , Hyaluronan Receptors/genetics , Mammary Glands, Animal/chemistry , Mammary Neoplasms, Experimental/chemistry , Rats , Receptors, Progesterone/analysis , Transforming Growth Factor beta1/geneticsABSTRACT
Cancer stem cells are resistant to current chemotherapy and radiation regimens available for breast cancer, making it imperative to study the mechanisms of resistance and development of therapeutic strategies that targets the tumor initiating cell population. One of the difficulties in identifying new drug targets has been that our current high throughput drug screens look for tumor shrinkage and do not incorporate the impact of compounds on the cancer stem cell population. In this review we discuss the literature on treatment resistance in breast cancer and the design of new clinical trials for test compounds which will allow us to determine both the reduction in tumor size and decrease in cancer stem cell population. In order to detect the effect of target compounds on cancer stem cells in a clinical setting, we will need to do multiple assays which include high throughput flow sorting analysis to determine the total number of CD44(+)/CD24(-/low)/Lin(-) and ALDH1 positive cells, as well as in-vitro mammosphere formation assay which is a functional assay dependent on the self renewal and anchorage independent growth properties of these cells.
Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/therapy , Drug Resistance, Neoplasm , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Animals , Clinical Trials as Topic , Humans , Neoplastic Stem Cells/metabolismABSTRACT
PURPOSE: The involvement of phosphatase and tensin homologue deleted on chromosome ten (PTEN) in endometrial carcinoma has implicated phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin (mTOR) activation in this disease. Understanding the extent of mTOR involvement and the mechanism responsible for activation is important, as mTOR inhibitors are currently being evaluated in clinical trials for endometrial carcinoma. Although tuberous sclerosis complex 2 (TSC2) is the "gatekeeper" for mTOR activation, little is known about defects in the TSC2 tumor suppressor or signaling pathways that regulate TSC2, such as LKB1/AMP-activated protein kinase, in the development of endometrial carcinoma. EXPERIMENTAL DESIGN: We determined the frequency of mTOR activation in endometrial carcinoma (primary tumors and cell lines) and investigated PTEN, LKB1, and TSC2 defects as underlying cause(s) of mTOR activation, and determined the ability of rapamycin to reverse these signaling defects in endometrial carcinoma cells. RESULTS: Activation of mTOR was a consistent feature in endometrial carcinomas and cell lines. In addition to PTEN, loss of TSC2 and LKB1 expression occurred in a significant fraction of primary tumors (13% and 21%, respectively). In tumors that retained TSC2 expression, phosphorylation of tuberin at S939 was observed with a high frequency, indicating that mTOR repression by TSC2 had been relieved via AKT phosphorylation of this tumor suppressor. In PTEN-null and LKB1-null endometrial carcinoma cell lines with functional inactivation of TSC2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 were able to inhibit AKT and mTOR signaling and reverse TSC2 phosphorylation. In contrast, although rapamycin inhibited mTOR signaling, it did not relieve phosphorylation of TSC2 at S939. CONCLUSIONS: Inactivation of TSC2 via loss of expression or phosphorylation occurred frequently in endometrial carcinoma to activate mTOR signaling. High-frequency mTOR activation supports mTOR as a rational therapeutic target for endometrial carcinoma. However, whereas rapamycin and its analogues may be efficacious at inhibiting mTOR activity, these drugs do not reverse the functional inactivation of TSC2 that occurs in these tumors.
Subject(s)
Endometrial Neoplasms/metabolism , PTEN Phosphohydrolase/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , AMP-Activated Protein Kinase Kinases , Androstadienes/pharmacology , Cell Line, Tumor , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Female , Humans , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tuberous Sclerosis , Tuberous Sclerosis Complex 2 Protein , WortmanninABSTRACT
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.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Docetaxel/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , omega-N-Methylarginine/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Docetaxel/therapeutic use , Drug Synergism , Female , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/drug effects , Mice , Mice, SCID , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor Assays , omega-N-Methylarginine/therapeutic useABSTRACT
Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.
Subject(s)
Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Receptors, Leptin/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Line, Tumor , Female , Humans , Mice , Mice, SCID , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasm Proteins/genetics , Neoplastic Stem Cells/pathology , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Receptors, Leptin/genetics , Response Elements , STAT3 Transcription Factor/genetics , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathologyABSTRACT
Induction of low-density lipoprotein (LDL) receptor transcription in response to depletion of cellular sterols in animal cells is well established. The intracellular signal or signals involved in regulating this process, however, remain unknown. Using a specific inhibitor of protein kinase C (PKC), calphostin C, we show the requirement of this kinase in the induction process in human hepatoma HepG2 cells. Overexpression of PKC epsilon, but not PKC alpha, -gamma, -delta, or -zeta was found to dramatically induce (approximately 18-fold) LDL receptor promoter activity. Interestingly, PKC epsilon-mediated induction was found to be sterol resistant. To further establish that PKC epsilon is involved in the sterol regulation of LDL receptor gene transcription, endogenous PKC epsilon was specifically inhibited by transfection with antisense PKC epsilon phosphorothionate oligonucleotides. Antisense treatment decreased endogenous PKC epsilon protein levels and completely blocked induction of LDL receptor transcription following sterol depletion. PKC epsilon-induced LDL receptor transcription is independent of the extracellular signal-regulated kinase 1 and 2 (p42/44(MAPK)) cascade, because the MEK-1/2 inhibitor, PD98059 did not inhibit, even though it blocked p42/44(MAPK) activation. Finally, photoaffinity labeling studies showed an isoform-specific interaction between PKC epsilon and sterols, suggesting that sterols may directly modulate its function by hampering binding of activators. This was confirmed by PKC activity assays. Altogether, these results define a novel signaling pathway leading to induction of LDL receptor transcription following sterol depletion, and a model is proposed to account for a new function for PKC epsilon as part of a sterol-sensitive signal transduction pathway in hepatic cells.
Subject(s)
Cholesterol/metabolism , Isoenzymes/metabolism , Protein Kinase C/metabolism , Receptors, LDL/biosynthesis , Receptors, LDL/genetics , Animals , Diglycerides/metabolism , Eicosanoids/metabolism , Gene Expression , Humans , Isoenzymes/genetics , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Phospholipids/metabolism , Protein Kinase C/genetics , Protein Kinase C-epsilon , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Transcription, Genetic , Tumor Cells, Cultured , Type C Phospholipases/metabolismABSTRACT
Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.