RESUMO
PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors. METHODS: We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment. RESULTS: In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets. CONCLUSIONS: Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.
Assuntos
Neoplasias de Mama Triplo Negativas , Negro ou Afro-Americano/genética , Feminino , Gana/epidemiologia , Humanos , Recidiva Local de Neoplasia , Neoplasias de Mama Triplo Negativas/genética , População BrancaRESUMO
INTRODUCTION: Interrogation of cancers with next-generation sequencing (NGS) mutation panels has become widely utilized, identifying prognostic and actionable mutations. This study explored the value of expanded mutation analysis in appendix peritoneal metastases (APM). METHODS: Forty-eight APM patients treated 2013-2018 were retrospectively collected from a registry. Fifty-gene NGS analysis was performed in CLIA approved lab to obtain mutation profiles. All patients underwent cytoreductive surgery (CRS)/hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C. Peritoneal cancer index (PCI), optimal CRS, survival (overall survival [OS] and progression-free survival [PFS]) data were collected. Survival analyses were performed on all APM, high-grade (HG), and low grade (LG) subsets, evaluating the impact of specific mutations on the outcome. RESULTS: Eighty-three percent of APM had a mutation identified. KRAS was most frequent, 65% (88% LG 42% HG) with GNAS identified in 92% of LG-APM. SMAD4 and/or TP53 mutations occurred in 25% of APM with observed decreased OS (46 vs. 81 months p = .0029); worse in HG-APM (26 vs. 49 months p = .0451). SMAD4 was associated with the most significant reduction in PFS in APM (p = .0085). Actionable mutations were identified in 73% of APM patients. CONCLUSIONS: Most frequent mutations were KRAS, TP53, and SMAD4, and actionable mutation detection was common. SMAD4 and TP53 were associated with decreased OS. NGS mutation profiling has potential utility in APM.
Assuntos
Neoplasias do Apêndice/genética , Neoplasias do Apêndice/terapia , Quimioterapia Intraperitoneal Hipertérmica/métodos , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Apêndice/patologia , Terapia Combinada , Procedimentos Cirúrgicos de Citorredução/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mitomicina/administração & dosagem , Neoplasias Peritoneais/secundário , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Mean arterial pressure (MAP), bispectral index (BIS), and minimum alveolar concentration (MAC) represent valuable, yet dynamic intraoperative monitoring variables. They provide information related to poor outcomes when considered together, however their collective behavior across time has not been characterized. METHODS: We have developed the Triple Variable Index (TVI), a composite variable representing the sum of z-scores from MAP, BIS, and MAC values that occur together during surgery. We generated a TVI expression profile, defined as the sequential TVI values expressed across time, for each surgery where concurrent MAP, BIS, and MAC monitoring occurred in an adult patient (≥18 years) at the University of Pittsburgh Medical Center between January and July 2014 (n = 5296). Patterns of TVI expression were identified using k-means clustering and compared across numerous patient, procedure, and outcome characteristics. TVI and the triple low state were compared as prediction models for 30-day postoperative mortality. RESULTS: The median frequency MAP, BIS, and MAC were recorded was one measurement every 3, 5, and 5 min. Three expression patterns were identified: elevated, mixed, and depressed. The elevated pattern displayed the highest average MAP, BIS, and MAC values (86.5 mmHg, 45.3, and 0.98, respectively), while the depressed pattern displayed the lowest values (76.6 mmHg, 38.0, 0.66). Patterns (elevated, mixed, depressed) were distinct across the following characteristics: average patient age (52, 53, 54 years), American Society of Anesthesiologists Physical Status 4 (6.7, 16.1, 27.3%) and 5 (0.1, 0.6, 1.6%) categories, cardiac (2.2, 6.5, 16.1%) and emergent (5.8, 10.5, 12.8%) surgery, cardiopulmonary bypass use (0.3, 2.6, 9.8%), intraoperative medication administration including etomidate (3.0, 7.3, 12.6%), hydromorphone (47.6, 26.3, 25.2%), ketamine (11.2, 4.6, 3.0%), dexmedetomidine (18.4, 16.6, 13.6%), phenylephrine (74.0, 74.8, 83.0), epinephrine (2.0, 6.0, 18.0%), norepinephrine (2.4, 7.5, 21.2%), vasopressin (3.4, 7.6, 21.0%), succinylcholine (74.0, 69.0, 61.9%), intraoperative hypotension (28.8, 33.0, 52.3%) and the triple low state (9.4, 30.3, 80.0%) exposure, and 30-day postoperative mortality (0.8, 2.7, 5.6%). TVI was a better predictor of patients that died or survived in the 30 days following surgery compared to cumulative triple low state exposure (AUC 0.68 versus 0.62, p < 0.05). CONCLUSIONS: Surgeries that share similar patterns of TVI expression display distinct patient, procedure, and outcome characteristics.
Assuntos
Pressão Arterial/fisiologia , Monitores de Consciência , Monitorização Intraoperatória/métodos , Alvéolos Pulmonares/fisiologia , Procedimentos Cirúrgicos Torácicos , Adulto , Ponte Cardiopulmonar/mortalidade , Humanos , Aprendizado de Máquina , Pessoa de Meia-Idade , Medicina PerioperatóriaRESUMO
Cancers are initiated and developed from a small population of stem-like cells termed cancer stem cells (CSCs). There is heterogeneity among this CSC population that leads to multiple subpopulations with their own distinct biological features and protein expression. The protein expression and function may be impacted by amino acid variants that can occur largely due to single nucleotide changes. We have thus performed proteomic analysis of breast CSC subpopulations by mass spectrometry to study the presence of single amino acid variants (SAAVs) and their relation to breast cancer. We have used CSC markers to isolate pure breast CSC subpopulation fractions (ALDH+ and CD44+/CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF-7 breast cancer cell line. By searching the Swiss-CanSAAVs database, 374 unique SAAVs were identified in total, where 27 are cancer-related SAAVs. 135 unique SAAVs were found in the CSC population compared with the mature luminal cells. The distribution of SAAVs detected in MCF-7 cells was compared with those predicted from the Swiss-CanSAAVs database, where we found distinct differences in the numbers of SAAVs detected relative to that expected from the Swiss-CanSAAVs database for several of the amino acids.
Assuntos
Aldeído Desidrogenase/genética , Substituição de Aminoácidos , Biomarcadores Tumorais/genética , Antígeno CD24/genética , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/metabolismo , Aldeído Desidrogenase/metabolismo , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Separação Celular , Bases de Dados de Proteínas , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Expressão Gênica , Ontologia Genética , Humanos , Receptores de Hialuronatos/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Células MCF-7 , Células-Tronco Neoplásicas/patologia , Mapeamento de Interação de ProteínasRESUMO
Curcumin is a potential agent for both the prevention and treatment of cancers. Curcumin treatment alone, or in combination with piperine, limits breast stem cell self-renewal, while remaining non-toxic to normal differentiated cells. We paired fluorescence-activated cell sorting with RNA sequencing to characterize the genome-wide changes induced specifically in normal breast stem cells following treatment with these compounds. We generated genome-wide maps of the transcriptional changes that occur in epithelial-like (ALDH+) and mesenchymal-like (ALDH-/CD44+/CD24-) normal breast stem/progenitor cells following treatment with curcumin and piperine. We show that curcumin targets both stem cell populations by down-regulating expression of breast stem cell genes including ALDH1A3, CD49f, PROM1, and TP63. We also identified novel genes and pathways targeted by curcumin, including downregulation of SCD. Transient siRNA knockdown of SCD in MCF10A cells significantly inhibited mammosphere formation and the mean proportion of CD44+/CD24- cells, suggesting that SCD is a regulator of breast stemness and a target of curcumin in breast stem cells. These findings extend previous reports of curcumin targeting stem cells, here in two phenotypically distinct stem/progenitor populations isolated from normal human breast tissue. We identified novel mechanisms by which curcumin and piperine target breast stem cell self-renewal, such as by targeting lipid metabolism, providing a mechanistic link between curcumin treatment and stem cell self-renewal. These results elucidate the mechanisms by which curcumin may act as a cancer-preventive compound and provide novel targets for cancer prevention and treatment.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Curcumina/farmacologia , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Estearoil-CoA Dessaturase/genética , Alcaloides/farmacologia , Benzodioxóis/farmacologia , Neoplasias da Mama/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Separação Celular , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem-like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label-free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24-, ALDH+ versus CD49f-EpCAM+ and CD44+CD24- versus CD49f-EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti-CSC therapeutics.
Assuntos
Neoplasias da Mama/química , Células-Tronco Neoplásicas/química , Proteoma/análise , Aldeído Desidrogenase/análise , Antígenos de Neoplasias/análise , Neoplasias da Mama/patologia , Antígeno CD24/análise , Moléculas de Adesão Celular/análise , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Receptores de Hialuronatos/análise , Integrina alfa6/análise , Células MCF-7 , Transdução de SinaisRESUMO
MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFß signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.
Assuntos
Neoplasias da Mama/genética , Diferenciação Celular/genética , Transformação Celular Neoplásica , MicroRNAs/genética , Células-Tronco Neoplásicas , Animais , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/metabolismo , Camundongos , MicroRNAs/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo , Neoplasias Experimentais , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genéticaRESUMO
Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34(+)CD38(-) AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias.
Assuntos
Adenosina/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Cinetina/uso terapêutico , Leucemia/tratamento farmacológico , Leucemia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Adenosina/análise , Adenosina/isolamento & purificação , Adenosina/farmacologia , Animais , Antineoplásicos/análise , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Cinetina/análise , Cinetina/isolamento & purificação , Cinetina/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: This study evaluates the utility of machine learning (ML) and natural language processing (NLP) in the processing and initial analysis of data within the electronic health record (EHR). We present and evaluate a method to classify medication names as either opioids or non-opioids using ML and NLP. Patients and Methods: A total of 4216 distinct medication entries were obtained from the EHR and were initially labeled by human reviewers as opioid or non-opioid medications. An approach incorporating bag-of-words NLP and supervised ML classification was implemented in MATLAB and used to automatically classify medications. The automated method was trained on 60% of the input data, evaluated on the remaining 40%, and compared to manual classification results. Results: A total of 3991 medication strings were classified as non-opioid medications (94.7%), and 225 were classified as opioid medications by the human reviewers (5.3%). The algorithm achieved a 99.6% accuracy, 97.8% sensitivity, 94.6% positive predictive value, F1 value of 0.96, and a receiver operating characteristic (ROC) curve with 0.998 area under the curve (AUC). A secondary analysis indicated that approximately 15-20 opioids (and 80-100 non-opioids) were needed to achieve accuracy, sensitivity, and AUC values of above 90-95%. Conclusion: The automated approach achieved excellent performance in classifying opioids or non-opioids, even with a practical number of human reviewed training examples. This will allow a significant reduction in manual chart review and improve data structuring for retrospective analyses in pain studies. The approach may also be adapted to further analysis and predictive analytics of EHR and other "big data" studies.
RESUMO
The nonobese diabetic/severe combined immune deficiency (NOD-scid) xenotransplantation model is the "gold standard" for assaying human hematopoietic stem cell activity. Systematic advancements, such as depletion of natural killer cell activity with anti-CD122 antibody, direct intrafemoral injection, and deletion or truncation of IL2Rgamma, have improved human cell engraftment; however, questions remain whether these mouse models are equivalent or, if not, which model is superior for assaying hematopoietic stem cell activity. To address this, we compared overall engraftment and multilineage differentiation of near-limiting doses of lineage-depleted human umbilical cord blood cells by direct intrafemoral injection into NOD/Lt-scid, NOD/Shi-scid, NOD/Lt-scid/IL2Rgamma(null) (NSG), and NOD/Shi-scid/IL2Rgamma(null) mice. Transplantation into NSG mice generated moderately higher human engraftment levels in bone marrow compared with other strains. At limiting doses, NSG mice of both sexes were 3.6-fold more sensitive in detecting SCID-repopulating cells compared with NOD/Lt-scid mice. However, NSG females exhibited higher engraftment at limiting cell doses, resulting in an overall increase in SCID-repopulating cell detection of 9-fold. Both NSG and NOD/Shi-scid/IL2Rgamma(null) support significantly improved engraftment in peripheral tissues compared with NOD/Lt-scid and NOD/Shi-scid mice, whereas NSG mice provide greater human engraftment in bone marrow than all other strains, especially at limiting doses.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Camundongos Endogâmicos NOD , Modelos Animais , Transplante Heterólogo/imunologia , Transplante Heterólogo/métodos , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Separação Celular , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , CamundongosRESUMO
Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds, we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia, myeloma, and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore, oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition, oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models, thereby establishing its ability to target leukemia stem cells. Mechanistically, CPX bound intracellular iron, and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance, CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus, in summary, CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore, CPX could be rapidly repurposed for the treatment of malignancies, including leukemia and myeloma.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Leucemia Mieloide Aguda/patologia , Mieloma Múltiplo/patologia , Piridonas/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Células Cultivadas , Ciclopirox , Citarabina/farmacologia , Sinergismo Farmacológico , Espectroscopia de Ressonância de Spin Eletrônica , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Luciferases/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Regiões Promotoras Genéticas , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo , SurvivinaRESUMO
For the past 20 years the mouse has served as a workhorse for studying the molecular underpinnings of human breast cancer. While some genetically engineered mouse mammary tumor models do not accurately recapitulate human disease (that is, the MMTV-Neu model and HER2-overexpressing human cancers), additional tumor models exist for studying the initiation, progression, and treatment of human breast cancer. The relationships between normal mouse mammary epithelial cells and tumor cells, however, have remained poorly defined. A recent article in Breast Cancer Research has shed light on these relationships.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Vírus do Tumor Mamário do Camundongo/genética , Animais , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Receptor ErbB-2RESUMO
Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.
Assuntos
Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have poor prognosis with less than 1-year median survival. Platinum-based chemotherapy remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSC exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to contribute to tumor recurrence. These head and neck CSCs (HNCSC) are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. Here, we performed microarray analysis in two HNSCC cell lines (UM-SCC-1, UM-SCC-22B) to investigate molecular pathways active in untreated and cisplatin-resistant ALDHhighCD44high cells. Gene set enrichment analysis and iPathway analysis identified signaling pathways with major implications to the pathobiology of cancer (e.g. TNFα, IFN, IL6/STAT, NF-κB) that are enriched in cisplatin-resistant ALDHhighCD44high cells, when compared to control cells. FGF2 was also enriched in cisplatin-resistant ALDHhighCD44high, which was confirmed by ELISA analysis. Inhibition of FGF signaling using BGJ398, a pan-FGF receptor (FGFR) small-molecule inhibitor, decreased ALDHhighCD44high alone in UM-SCC-1 and preferentially targeted cisplatin-resistant ALDHhighCD44high cells in UM-SCC-22B. These findings suggest that FGFR signaling might play an important role in the resistance of head and neck CSC to cisplatin. Collectively, this work suggests that some head and neck cancer patients might benefit from the combination of cisplatin and a FGFR inhibitor.
RESUMO
During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/- or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH-CD44+CD24- human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24- by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity.
Assuntos
Mama/citologia , Perfilação da Expressão Gênica , Células-Tronco/metabolismo , Aldeído Desidrogenase/metabolismo , Biomarcadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Sobrevivência Celular , Epitélio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Receptores de Hialuronatos/metabolismo , Mesoderma/metabolismo , Fenótipo , Transcriptoma/genéticaRESUMO
Triple negative breast cancer (TNBC) typically exhibits rapid progression, high mortality and faster relapse rates relative to other breast cancer subtypes. In this report we examine the combination of taxanes (paclitaxel or docetaxel) with a breast cancer stem cell (CSC)-targeting agent sulforaphane for use against TNBC. We demonstrate that paclitaxel or docetaxel treatment induces IL-6 secretion and results in expansion of CSCs in TNBC cell lines. Conversely, sulforaphane is capable of preferentially eliminating CSCs, by inhibiting NF-κB p65 subunit translocation, downregulating p52 and consequent downstream transcriptional activity. Sulforaphane also reverses taxane-induced aldehyde dehydrogenase-positive (ALDH+) cell enrichment, and dramatically reduces the size and number of primary and secondary mammospheres formed. In vivo in an advanced treatment orthotopic mouse xenograft model together with extreme limiting dilution analysis (ELDA), the combination of docetaxel and sulforaphane exhibits a greater reduction in primary tumor volume and significantly reduces secondary tumor formation relative to either treatment alone. These results suggest that treatment of TNBCs with cytotoxic chemotherapy would be greatly benefited by the addition of sulforaphane to prevent expansion of and eliminate breast CSCs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Isotiocianatos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Paclitaxel/farmacologia , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Sulfóxidos , Fatores de Tempo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because cancer stem cells (CSCs) have been implicated in chemo-resistance, metastasis and tumor recurrence, therapeutic targeting of CSCs holds promise to address these clinical challenges to cancer treatment. VS-4718 and VS-6063 are potent inhibitors of focal adhesion kinase (FAK), a non-receptor tyrosine kinase that mediates cell signals transmitted by integrins and growth factor receptors. We report here that inhibition of FAK kinase activity by VS-4718 or VS-6063 preferentially targets CSCs, as demonstrated by a panel of orthogonal CSC assays in cell line models and surgically resected primary breast tumor specimens cultured ex vivo. Oral administration of VS-4718 or VS-6063 to mice bearing xenograft models of triple-negative breast cancer (TNBC) significantly reduced the proportion of CSCs in the tumors, as evidenced by a reduced tumor-initiating capability upon re-implantation in limiting dilutions of cells prepared from these tumors. In contrast, the cytotoxic chemotherapeutic agents, paclitaxel and carboplatin, enriched for CSCs, consistent with previous reports that these cytotoxic agents preferentially target non-CSCs. Importantly, VS-4718 and VS-6063 attenuated the chemotherapy-induced enrichment of CSCs in vitro and delayed tumor regrowth following cessation of chemotherapy. An intriguing crosstalk between FAK and the Wnt/ß-catenin pathway was revealed wherein FAK inhibition blocks ß-catenin activation by reducing tyrosine 654 phosphorylation of ß-catenin. Furthermore, a constitutively active mutant form of ß-catenin reversed the preferential targeting of CSCs by FAK inhibition, suggesting that this targeting is mediated, at least in part, through attenuating ß-catenin activation. The preferential targeting of cancer stem cells by FAK inhibitors provides a rationale for the clinical development of FAK inhibitors aimed to increase durable responses for cancer patients.
RESUMO
The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Processos de Crescimento Celular/fisiologia , Dano ao DNA/fisiologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
INTRODUCTION: Determination of fine-needle aspiration (FNA) material adequacy is essential prior to performing molecular testing (MT) in order to ensure good results and maximize resources. This study investigates several quantitative measures of cellularity in FNA samples of lung carcinoma, and correlates the results with MT adequacy. MATERIALS AND METHODS: A blinded retrospective analysis of 20 non-small-cell lung carcinoma cases was conducted: 13 contained "sufficient" material for EGFR/KRAS sequencing and ALK FISH studies; and 7 contained "insufficient" material for these tests. Three 400x fields-of-view (FOVs) were analyzed from digitized cell block glass slides of these cases. Cellularity in these FOVs was quantified using three methods: (1) visual estimation by cytopathologist; (2) manually annotated contours (MACs); (3) software derived, manually adjusted contours (SDMACs) using a custom segmentation script with adjustable parameters. These methods were evaluated using the Mann-Whitney-Wilcoxon test, paired t test, and receiver operating characteristic/area under the curve (AUC) analysis. RESULTS: There were significant differences between the insufficient/sufficient groups for each estimation method (visual P < 0.05, MAC P < 0.05, SDMAC P < 0.01). Variation of mean values was highest in the visual estimation method. AUC values were visual estimation = 0.903, MAC = 0.903, and SDMAC = 0.958. Mean variation of the 3 FOV values was found to be significantly higher for visual estimation compared with the other methods. CONCLUSION: Quantitative analysis of cellularity in digitized cell block material is feasible using different methods. In this investigation, the SDMAC method provided the highest accuracy and lowest variability. This supports image analysis as an objective and quantitative tool to assess FNA sample adequacy for guiding supplemental MT.
RESUMO
Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.