RESUMEN
BACKGROUND: There are profound individual differences in clinical outcomes between colorectal cancers (CRCs) presenting with identical stage of disease. Molecular stratification, in conjunction with the traditional TNM staging, is a promising way to predict patient outcomes. We investigated the interconnectivity between tumor stage and tumor biology reflected by the Consensus Molecular Subtypes (CMSs) in CRC, and explored the possible value of these insights in patients with stage II colon cancer. METHODS: We performed a retrospective analysis using clinical records and gene expression profiling in a meta-cohort of 1040 CRC patients. The interconnectivity of tumor biology and disease stage was assessed by investigating the association between CMSs and TNM classification. In order to validate the clinical applicability of our findings we employed a meta-cohort of 197 stage II colon cancers. RESULTS: CMS4 was significantly more prevalent in advanced stages of disease (stage I 9.8% versus stage IV 38.5%, p < 0.001). The observed differential gene expression between cancer stages is at least partly explained by the biological differences as reflected by CMS subtypes. Gene signatures for stage III-IV and CMS4 were highly correlated (r = 0.77, p < 0.001). CMS4 cancers showed an increased progression rate to more advanced stages (CMS4 compared to CMS2: 1.25, 95% CI: 1.08-1.46). Patients with a CMS4 cancer had worse survival in the high-risk stage II tumors compared to the total stage II cohort (5-year DFS 41.7% versus 100.0%, p = 0.008). CONCLUSIONS: Considerable interconnectivity between tumor biology and tumor stage in CRC exists. This implies that the TNM stage, in addition to the stage of progression, might also reflect distinct biological disease entities. These insights can potentially be utilized to optimize identification of high-risk stage II colon cancers.
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Neoplasias del Colon/genética , Neoplasias del Colon/patología , Transcriptoma , Anciano , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , RiesgoRESUMEN
The CC-chemokine receptor CCR5 has been associated with cancer progression and metastasis. CCR5 blockers such as Maraviroc are tested in metastatic cancer patients. A mutant allele of CCR5, CCR5-delta32 (CCR5del32), which encodes for a protein with a trans-dominant negative effect on the wildtype protein, is frequently found in populations of northern European origin. We set out to determine if the CCR5del32 genotype is associated with progression of breast cancer. Here, we genotyped 414 breast cancer patients and investigated whether the CCR5 genotype had an association with the likelihood to metastasize within specific subgroups of this cohort. The findings were subsequently confirmed in an independent cohort of 1,017 breast cancer patients. Specifically within the postmenopausal subgroup of the initial cohort (n = 325) individuals carrying the CCR5del32 genotype exhibited a significantly longer metastasis-free survival (MFS, p = 0.038). In an independent cohort, CCR5del32 genotype was confirmed to be associated with prolonged MFS only in postmenopausal patients (n = 579, hazard ratio [HR] = 0.61, 95% confidence interval [95% CI] = 0.38-0.99, p = 0.044), and not in premenopausal patients (n = 438, HR = 1.01, 95% CI = 0.70-1.48, p = 0.94). Our results indicate that CCR5del32 genotype is associated with good prognosis in postmenopausal breast cancer patients. Considering this result, postmenopausal breast cancer patients who are wildtype for CCR5 genotype might benefit from CCR5 blockers, such as Maraviroc.
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Neoplasias de la Mama/genética , Mutación del Sistema de Lectura , Receptores CCR5/genética , Adolescente , Adulto , Anciano , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Niño , Preescolar , Análisis Mutacional de ADN , Supervivencia sin Enfermedad , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Recién Nacido , Estimación de Kaplan-Meier , Persona de Mediana Edad , Posmenopausia , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Eliminación de Secuencia , Adulto JovenRESUMEN
BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Perfilación de la Expresión Génica/métodos , Células Neoplásicas Circulantes , Adulto , Bélgica/epidemiología , Neoplasias de la Mama/epidemiología , Estudios de Cohortes , Femenino , Humanos , Persona de Mediana Edad , Países Bajos/epidemiología , Pronóstico , Estudios ProspectivosRESUMEN
BACKGROUND: We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy. METHODS: One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy. RESULTS: The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59-6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79-3.93) and 2.05 (1.41-2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed. CONCLUSIONS: The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Quinasa de Punto de Control 2/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Quimioterapia Adyuvante/métodos , Supervivencia sin Enfermedad , Femenino , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Mutación/genéticaRESUMEN
BACKGROUND: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation. METHODS: Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis. RESULTS: On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines. CONCLUSION: We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.
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Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Separación Celular/métodos , Perfilación de la Expresión Génica , Células Neoplásicas Circulantes/patología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Separación Celular/instrumentación , Femenino , Humanos , Mutación/genéticaRESUMEN
BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Moduladores de los Receptores de Estrógeno/uso terapéutico , Terapia Molecular Dirigida/métodos , Quinazolinas/uso terapéutico , ARN no Traducido/metabolismo , Receptor ErbB-2/metabolismo , Tamoxifeno/uso terapéutico , Adulto , Anciano , Antineoplásicos/farmacología , Antineoplásicos Hormonales/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/mortalidad , Proliferación Celular , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/farmacología , ARN Largo no Codificante , ARN Mensajero/metabolismo , ARN no Traducido/efectos de los fármacos , ARN no Traducido/genética , Receptor ErbB-2/efectos de los fármacos , Receptor ErbB-2/genética , Receptores de Estrógenos/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.
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Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Ciclina B/genética , Proteínas de Unión al ADN/genética , MicroARNs/genética , Tamoxifeno/uso terapéutico , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Proteína Quinasa CDC2 , Ciclina E/genética , Quinasas Ciclina-Dependientes , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Oncogénicas/genética , Complejo Represivo Polycomb 2 , Transducción de Señal , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.
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Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/genética , Tamoxifeno/uso terapéutico , Factores de Transcripción/genética , Antineoplásicos Hormonales/farmacología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Estradiol/análogos & derivados , Estradiol/farmacología , Estradiol/uso terapéutico , Moduladores de los Receptores de Estrógeno/farmacología , Moduladores de los Receptores de Estrógeno/uso terapéutico , Receptor alfa de Estrógeno/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Silenciador del Gen , Humanos , Metástasis de la Neoplasia , Complejo Represivo Polycomb 2 , Reacción en Cadena de la Polimerasa , Alcamidas Poliinsaturadas/farmacología , Alcamidas Poliinsaturadas/uso terapéutico , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño , Tamoxifeno/farmacología , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: Cisplatin (cDDP) has regained interest for metastatic breast cancer (MBC) patients, given the platinum sensitivity in subtypes and better manageable toxicity. Here, the primary aim was to determine whether molecular characteristics of circulating tumor cells (CTCs) could identify patients responding to cDDP and to describe the outcomes to cDDP monotherapy in a large group of MBC patients pretreated with anthracycline- and taxane-based treatments. METHODS: Based on cell line data, a CTC-cDDP-sensitivity profile was generated. Applying an A'Herns single-stage phase II design, further investigation was considered worthwhile if 5/10 patients with a favorable profile responded to cDDP. Patients received 70mg/m2 cDDP every three weeks, CTCs were enumerated and the CTC-cDDP-sensitivity profile was determined. In total, 65 heavily pretreated MBC patients (77% received ≥2 lines of previous chemotherapy for MBC) were eligible for the per-protocol analysis. Primary endpoint was response rate, secondary endpoints included best observed response, progression-free survival (PFS) and overall survival (OS). RESULTS: The best observed response during cDDP therapy was a partial response in 7% and stable disease in 56% of the patients. None of the patients with a favorable CTC-cDDP-sensitivity profile had a response. The median baseline CTC count was 8 (range 0-3254). Patients with <5 CTCs had a better PFS and OS than patients with ≥5 CTCs (median PFS 4.5 months (95%CI 2.38-6.62) vs. 2.1 months [(95%CI 1.34-2.80)(p=0.009)] and median OS 13.1 months (95%CI 9.89-16.33) vs. 5.6 months [(95%CI 3.60-7.64)(p=0.003)]. No other factors than CTC count were associated with outcome to cDDP therapy, including triple-negative breast cancer versus ER-positive tumors. CONCLUSIONS: The CTC-cDDP-sensitivity profile was unable to select patients responding to cDDP monotherapy. In an unselected group of heavily pretreated MBC patients, cDDP yields outcomes comparable to other chemotherapeutic regimens for heavily pretreated MBC patients. CTC count was the only factor associated with outcome in these patients. CLINICAL TRIAL REGISTRATION: (https://www.trialregister.nl/trial/3885, identifier NTR4046).
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BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Proteína Sustrato Asociada a CrK/fisiología , Resistencia a Antineoplásicos/genética , Tamoxifeno/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Adulto , Anciano , Antineoplásicos Hormonales/uso terapéutico , Biomarcadores Farmacológicos/análisis , Biomarcadores Farmacológicos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Carcinoma/genética , Carcinoma/mortalidad , Línea Celular Tumoral , Proteína Sustrato Asociada a CrK/genética , Proteína Sustrato Asociada a CrK/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , ARN Largo no Codificante , ARN no Traducido , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
BACKGROUND: Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance. METHODS: Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer. RESULTS: mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease. CONCLUSIONS: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.
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Neoplasias de la Mama/metabolismo , Antagonistas de Estrógenos/farmacología , Co-Represor 2 de Receptor Nuclear/metabolismo , Proteínas Represoras/metabolismo , Tamoxifeno/farmacología , Transactivadores/metabolismo , Adulto , Anciano , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Persona de Mediana Edad , Co-Represor 2 de Receptor Nuclear/biosíntesis , Co-Represor 2 de Receptor Nuclear/genética , Receptores de Estrógenos/biosíntesis , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Transactivadores/biosíntesis , Transactivadores/genéticaRESUMEN
The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCdelta. Currently, it is unclear whether PKCdelta is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCdelta in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cdelta expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCdelta but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCdelta concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCdelta mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cdelta mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann-Whitney U-test). Increasing concentrations of PKCdelta mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCdelta in breast cancer progression.
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Western Blotting , Neoplasias de la Mama/metabolismo , Ensayo de Inmunoadsorción Enzimática , Proteína Quinasa C-delta/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Mama/genética , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteína Quinasa C-delta/genética , ARN Mensajero , Análisis de SupervivenciaRESUMEN
The large number of non-coding RNAs (ncRNAs) and their breadth of functionalities has fuelled many studies on their roles in cancer. We previously linked four microRNAs to breast cancer prognosis. One of these microRNAs, hsa-miR-7, was found to be regulated by another type of ncRNA, the circular non-coding RNA (circRNA) CDR1-AS, which contains multiple hsa-miR-7 binding sites. Based on this finding, we studied the potential clinical value of this circRNA on breast cancer prognosis in a cohort based on a cohort that was previously analysed for hsa-miR-7 and in an adjuvant hormone-naïve cohort for 1st-line tamoxifen treatment outcomes, in which we also analysed hsa-miR-7. A negative correlation was observed between hsa-miR-7 and CDR1-AS in both cohorts. Despite associations with various clinical metrics (e.g., tumour grade, tumour size, and relapse location), CDR1-AS was neither prognostic nor predictive of relevant outcomes in our cohorts. However, we did observe stromal CDR1-AS expression, suggesting a possible cell-type specific interaction. Next to the known association of hsa-miR-7 expression with poor prognosis in primary breast cancer, we found that high hsa-miR-7 expression was predictive of an adverse response to tamoxifen therapy and poor progression-free and post-relapse overall survival in patients with recurrent disease.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Tamoxifeno/uso terapéutico , Adulto , Anciano , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Largo no Codificante/metabolismo , RecurrenciaRESUMEN
About 70-80% of breast cancers express estrogen receptor alpha (ER-alpha), and estrogens play important roles in the development and growth of hormone-dependent tumors. Together with lymph node metastasis, tumor size, and histological grade, ER status is considered as one of the prognostic factors in breast cancer, and an indicator for hormonal treatment. To investigate genes and pathways that are associated with ER status and epithelial cells in breast tumor, we applied laser capture microdissection (LCM) technology to capture epithelial tumor cells from 28 lymph node-negative breast tumor samples, in which 17 patients had ER-alpha+ tumors, and 11 patients have ER-alpha- tumors. Gene expression profiles were analysed on Affymetrix Hu133A GeneChip. Meanwhile, gene profiles using total RNA isolated from bulk tumors of the same 28 patients were also generated. In total, 146 genes and 112 genes with significant P-value and having significant differential expression between ER-alpha+ and ER-alpha- tumors were identified from the LCM data set and bulk tissue data set, respectively. A total of 61 genes were found to be common in both data sets, while 85 genes were unique to the LCM data set and 51 genes were present only in the bulk tumor data set. Pathway analysis with the 85 genes using Gene Ontology suggested that genes involved in endocytosis, ceramide generation, Ras/ERK/Ark cascade, and JAT-STAT pathways may play roles related to ER. The gene profiling with LCM-captured tumor cells provides a unique approach to study epithelial tumor cells and to gain an insight into signaling pathways associated with ER.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Perfilación de la Expresión Génica , Receptores de Estrógenos/genética , Células Epiteliales , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Rayos Láser , Microdisección , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Estrógenos/análisis , Receptores de Estrógenos/biosíntesis , Transducción de SeñalRESUMEN
Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.
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Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Neoplasias Pulmonares/genética , Proto-Oncogenes/genética , Factor de Transcripción SOX9/genética , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Células MCF-7 , Proteína del Locus del Complejo MDS1 y EV11 , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Osteonectina/genética , Proteína Reguladora Asociada a mTOR , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Identification of specific risk groups for recurrence after surgery for isolated colorectal liver metastases (CRLM) remains challenging due to the heterogeneity of the disease. Classical clinicopathologic parameters have limited prognostic value. The aim of this study was to identify a gene expression signature measured in CRLM discriminating early from late recurrence after partial hepatectomy. METHODS: CRLM from two patient groups were collected: I) with recurrent disease ≤12 months after surgery (N = 33), and II) without recurrences and disease free for ≥36 months (N = 30). The patients were clinically homogeneous; all had a low clinical risk score (0-2) and did not receive (neo-) adjuvant chemotherapy. Total RNA was hybridised to Illumina arrays, and processed for analysis. A leave-one-out cross validation (LOOCV) analysis was performed to identify a prognostic gene expression signature. RESULTS: LOOCV yielded an 11-gene profile with prognostic value in relation to recurrent disease ≤12 months after partial hepatectomy. This signature had a sensitivity of 81.8%, with a specificity of 66.7% for predicting recurrences (≤12 months) versus no recurrences for at least 36 months after surgery (X2 P < 0.0001). CONCLUSION: The current study yielded an 11-gene signature at mRNA level in CRLM discriminating early from late or no relapse after partial hepatectomy.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/secundario , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Hígado/patología , ARN Mensajero/genética , Transcriptoma , Anciano , Biomarcadores de Tumor/genética , Colon/patología , Neoplasias Colorrectales/diagnóstico , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Hepatectomía , Humanos , Hígado/cirugía , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Pronóstico , Recto/patologíaRESUMEN
The Boyden chamber assay is widely used for in vitro measurement of the invasive capacity of cells. However, results can be affected significantly if certain precautions are not taken. Using the Boyden chamber assay we investigated in vitro the invasive potential of a variety of human gynecological tumor cell lines to degrade and migrate through the artificial basement membrane matrix Matrigel. However, in the absence of this Matrigel layer large differences were observed in the ability of cells to adhere to, migrate through and attach to the lower side of the filter membranes. These differences were influenced by cell density, degree of directional locomotion, and the size of the filter pores. To adjust for these influences (which are not directly correlated to the capacity of cells to traverse the Matrigel layer), invasion results were corrected for the ability of cells to migrate through the filter membrane. In addition, the invasion of MDA-MB-231 cells was used as an internal standard to compensate for variations in the Matrigel layer between different experiments. Overall, in our experimental set up, the five human breast cancer cell lines were the most invasive (mean invasion +/- SEM relative to MDA-MB-231 invasion: 104.7 +/- 6.1%), the five human ovarian cancer cell lines the least invasive (60.2 +/- 2.2%) and the six human endometrial cancer cell lines showed an intermediate capacity (79.1 +/- 3.5%). In conclusion, the Boyden chamber assay can be used reliably for studying the invasive potential of cells in vitro, if the ability of the cells to migrate through the filter is taken into account, and a reference cell line is included to enable comparison of the data obtained from independently performed experiments on different cell lines.
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Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula/instrumentación , Neoplasias de los Genitales Femeninos/patología , Invasividad Neoplásica , Células 3T3/metabolismo , Animales , Recuento de Células , Movimiento Celular , Tamaño de la Célula , Colágeno/metabolismo , Colágeno/farmacología , Medios de Cultivo Condicionados/farmacología , Combinación de Medicamentos , Neoplasias Endometriales/patología , Femenino , Fibronectinas/metabolismo , Fibronectinas/farmacología , Filtración/instrumentación , Humanos , Laminina/metabolismo , Laminina/farmacología , Membranas Artificiales , Ratones , Neoplasias Ováricas/patología , Cemento de Policarboxilato , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Células Tumorales CultivadasRESUMEN
Human breast fibroblasts have been shown to express urokinase-type plasminogen activator (uPA). This suggests that fibroblasts are actively involved in the process of uPA-directed breast tumor proteolysis. To investigate a possible role for the insulin-like growth factors (IGFs) in regulating uPA expression in human breast fibroblasts, we correlated the expression of uPA with the expression of IGF-1 and IGF-2 in a paired panel of normal and tumor tissue-derived human breast fibroblasts in vitro. Analysis of reverse transcribed polymerase chain reaction (RT-PCR) amplified mRNA revealed that the tumor-derived fibroblast strain expressed significantly more basal uPA mRNA and significantly less IGF-1 mRNA when compared to their normal counterpart. The expression of basal IGF-2 mRNA did not differ between these cultures. For both normal and tumor tissue-derived fibroblasts, cytokine- and growth factor-induced steady-state levels of uPA and IGF-1 mRNA were inversely related. No such correlation was found for uPA and IGF-2 mRNA. While exogenously added IGF-1 decreased the amount of uPA mRNA transcripts similarly in both normal and tumoral fibroblasts, exogenously added uPA decreased the amount of IGF-1 mRNA transcripts only in tumor tissue-derived fibroblasts. These data suggest that in human breast fibroblasts IGF-1 controls the expression of uPA and that, possibly due to an altered sensitivity to uPA, tumor-associated fibroblasts have escaped this local control mechanism.
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Mama/química , Fibroblastos/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Mama/citología , Células Cultivadas , Citocinas/farmacología , Femenino , Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , ARN Mensajero/efectos de los fármacos , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
In vitro exposure of estrogen receptor-negative (ER-) EVSA-T human breast cancer cells to insulin and/or estradiol had no effect on cell cycle distribution, in contrast to a 3-5-fold increase in the percentages of cells in the S-phase of the cell cycle in the ER+ MCF-7 cell line. Estrogen pretreatment of MCF-7 cells followed by incubation with doxorubicin resulted in an augmented inhibition of cell growth compared to unstimulated controls. This delay in growth was accompanied by a decrease in the percentages of cells actively synthesizing DNA, and by an augmented percentage of cells exhibiting a G2M-amount of DNA at the end of a 6-9 day period of culture in complete growth medium.
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Neoplasias de la Mama/tratamiento farmacológico , Ciclo Celular , Doxorrubicina/farmacología , Bromodesoxiuridina , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , ADN/metabolismo , Estradiol/farmacología , Femenino , Citometría de Flujo , Humanos , Insulina/farmacología , Cinética , Receptores de Estrógenos/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
UNLABELLED: Interference in growth factor mediated pathways is a new strategy in the treatment of cancer. Somatostatin analogs can inhibit hormone and growth factor secretion, while suramin can block the binding of several growth factors to their receptors. In addition, somatostatin analogs can cause direct growth inhibitory effects after binding to tumoral somatostatin receptors. We tested the efficacy and endocrine effects of chronic treatment with three somatostatin analogs (Sandostatin, R RC-160 and CGP 15-425) or suramin in several tumor models and in patients with various types of cancer. Treatment with somatostatin analogs caused growth inhibition of breast cancer cells (MCF-7) in vitro, and of rat transplantable pancreatic (50-70% inhibition) and prostatic Dunning tumors (12% inhibition). No tumor growth inhibition was observed with respect to DMBA-induced rat mammary tumors, a transplantable colon tumor and a rhabdomyosarcoma in rats. In 34 patients with metastatic pancreatic or gastrointestinal adenocarcinomas chronic Sandostatin treatment caused stable disease in 27% of the patients, but no objective remissions. Somatostatin receptors were found in the responding MCF-7 mammary tumor cells, rat pancreatic tumors and in 20-45% of human breast cancer specimens [J. Steroid Biochem. Molec. Biol. 37 (1990) 1073-1077], but not in rat DMBA-mammary tumors or in 10 human pancreatic adenocarcinomas. Suramin caused significant dose-dependent growth inhibition of human breast cancer cells in vitro and of rat pancreatic tumors in vivo in the presence of plasma levels up to 150 micrograms/ml. In a preliminary clinical study concerning 11 patients with various tumor types we observed significant hematological, biochemical, endocrine and clinical side effects, but no objective remissions in spite of relevant peak plasma suramin concentrations of 270-330 micrograms/ml. IN CONCLUSION: somatostatin analogs and suramin can cause growth inhibition of various experimental tumors in vitro and in vivo, but the clinical value has to be established for several types of cancer, especially with respect to suramin and suramin-like compounds.