RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease. For patients with localized PDAC, surgery is the best option, but with a median survival of less than 2 years and a difficult and prolonged postoperative course for most, there is an urgent need to better identify patients who have the most aggressive disease. METHODS AND FINDINGS: We analyzed the gene expression profiles of primary tumors from patients with localized compared to metastatic disease and identified a six-gene signature associated with metastatic disease. We evaluated the prognostic potential of this signature in a training set of 34 patients with localized and resected PDAC and selected a cut-point associated with outcome using X-tile. We then applied this cut-point to an independent test set of 67 patients with localized and resected PDAC and found that our signature was independently predictive of survival and superior to established clinical prognostic factors such as grade, tumor size, and nodal status, with a hazard ratio of 4.1 (95% confidence interval [CI] 1.7-10.0). Patients defined to be high-risk patients by the six-gene signature had a 1-year survival rate of 55% compared to 91% in the low-risk group. CONCLUSIONS: Our six-gene signature may be used to better stage PDAC patients and assist in the difficult treatment decisions of surgery and to select patients whose tumor biology may benefit most from neoadjuvant therapy. The use of this six-gene signature should be investigated in prospective patient cohorts, and if confirmed, in future PDAC clinical trials, its potential as a biomarker should be investigated. Genes in this signature, or the pathways that they fall into, may represent new therapeutic targets. Please see later in the article for the Editors' Summary.
Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/mortalidade , Perfilação da Expressão Gênica , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidade , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Metástase Neoplásica , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , Análise de Sobrevida , Análise Serial de TecidosRESUMO
BACKGROUND: Standard therapy for stage II/III rectal cancer consists of a fluoropyrimidine and radiation therapy followed by surgery. Preclinical data demonstrated that bortezomib functions as a radiosensitizer in colorectal cancer models. The purpose of this study was to determine the maximum tolerated dose (MTD) of bortezomib in combination with chemotherapy and radiation. PATIENTS AND METHODS: Patients with locally advanced rectal adenocarcinomas, as staged by endoscopic ultrasound, were eligible. Bortezomib was administered on days 1, 4, 8, and 11 every 21 days for 2 cycles with 5-fluorouracil at 225 mg/m2/day continuously and 50.4 Gy of radiation. Dose escalation of bortezomib was conducted via a standard 3 + 3 dose escalation design. A subset of patients underwent serial tumor biopsies for correlative studies. RESULTS: Nine patients in 2 dose cohorts were enrolled. Diarrhea was the principal dose-limiting toxicity and occurred at the 1.0-mg/m2 dose level. There was no clear evidence of suppression of nuclear factor-kappaB target gene expression in biopsy samples. CONCLUSION: The MTD of bortezomib in combination with chemotherapy and radiation may be below a clinically relevant dose, limiting the clinical applicability of this combination. Performing biopsies before and during irradiation for determining gene expression in response to radiation therapy is feasible.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Ácidos Borônicos/uso terapêutico , Fluoruracila/uso terapêutico , Pirazinas/uso terapêutico , Neoplasias Retais/tratamento farmacológico , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/radioterapia , Adulto , Idoso , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/uso terapêutico , Antineoplásicos/efeitos adversos , Ácidos Borônicos/efeitos adversos , Bortezomib , Progressão da Doença , Quimioterapia Combinada , Feminino , Fluoruracila/efeitos adversos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Pirazinas/efeitos adversos , Neoplasias Retais/mortalidade , Neoplasias Retais/patologia , Neoplasias Retais/radioterapiaRESUMO
In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80(HER4)) and subsequently liberates a soluble 80-kDa fragment, s80(HER4). Here we report that s80(HER4) Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80(HER4), the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80(HER4), a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80(HER4) degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80(HER4) is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.
Assuntos
Receptores ErbB/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Chlorocebus aethiops , Estabilidade Enzimática , Receptores ErbB/química , Regulação da Expressão Gênica , Humanos , Camundongos , Peso Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-4 , Solubilidade , Frações Subcelulares/enzimologia , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ErbB4 is often associated with growth-inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intramembraneous gamma-secretase, releasing the soluble, intracellular 80-kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacological inhibition of either gamma-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676-1308], GFP-CT(HER4) (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP-s80HER4 nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT)5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP-CTHER4 or GFP alone. The s80HER4-induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP-CTHER4-, and GFP-s80HER4-expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent beta-casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition.
Assuntos
Proliferação de Células , Citoplasma/fisiologia , Receptores ErbB/fisiologia , Inibidores do Crescimento/fisiologia , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Fragmentos de Peptídeos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Linhagem Celular Tumoral , Feminino , Humanos , Glândulas Mamárias Humanas/enzimologia , Neuregulina-1/antagonistas & inibidores , Neuregulina-1/fisiologia , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína , Receptor ErbB-4RESUMO
HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G(2)/M progression of breast cancer cells. While investigating pathways of G(2)/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.
Assuntos
Proteína BRCA1/metabolismo , Receptores ErbB/metabolismo , Mitose/efeitos dos fármacos , Neuregulina-1/farmacologia , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Neoplasias da Mama/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/genética , Éxons/genética , Fase G2/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-4 , Células Tumorais CultivadasRESUMO
PURPOSE: Efforts to conclusively establish that human epidermal receptor (HER)-2 overexpression is important to androgen-dependent carcinoma of the prostate (AD-CaP) or to progression to androgen independence (AI-CaP) have failed because of variability in tissue procurement, antibodies, immunostaining procedures, and assessment methods. However, because some in vitro and animal model data correlate HER-2 overexpression with progression to androgen independence, trials of agents that target the HER-2 receptor are under way. To clarify human tumor findings, we studied HER-2 expression at the gene (DNA), mRNA, and protein levels in well-characterized CaP specimens. EXPERIMENTAL DESIGN: Fifty AD-CaP and 25 AI-CaP specimens from similar numbers of Caucasian and African Americans were immunostained for HER-2 receptor. HER-2 mRNA levels were measured using real-time fluorescence quantitative PCR in patients for whom frozen specimens were available. HER-2 amplification was evaluated using fluorescent in situ hybridization. RESULTS: HER-2 receptor immunostained in 52% of androgen-dependent and one (4%) androgen-independent tumor. HER-2 immunostaining was not related to age, race, serum prostate-specific antigen levels, or pathologic stage and Gleason grade. HER-2 overexpression was not detected in AI-CaP at the mRNA or gene level. Mean HER-2 mRNA expression was higher (P < 0.05) in AD-CaP than AI-CaP (22,080 versus 15,496 HER-2 copies). HER-2 was not amplified in any of 20 AD-CaP or 19 AI-CaP specimens. CONCLUSIONS: HER-2 protein and message overexpression and HER-2 amplification were not found in AI-CaP.
Assuntos
Carcinoma/metabolismo , Genes erbB-2/genética , Neoplasias da Próstata/metabolismo , Receptor ErbB-2/biossíntese , Androgênios/metabolismo , Progressão da Doença , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Ligantes , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptor ErbB-2/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To determine expression levels of annexin I (lipocortin I) in patient-matched benign prostatic epithelium (BPE), high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate cancer (CaP). EXPERIMETNAL DESIGN: Annexin I protein expression was examined with a standard immunohistochemical protocol in 69 radical prostatectomy specimens, 45 of which also contained HGPIN. Immunostained sections were scored visually by a genitourinary pathologist and mean optical density was measured with digital image analysis. Real-time fluorescence quantitative PCR was used to measure expression levels of annexin I mRNA in patient-matched CaP and BPE from 14 snap-frozen, radical prostatectomy specimens. RESULTS: Annexin I protein expression was reduced in 91% (41/45) of HGPIN lesions and 94% (65/69) of invasive CaP compared with BPE in the same histological section when assessed visually. Mean absorbance was reduced significantly (P < 0.05) in 97.7% (44/45) of HGPIN lesions and 98.5% (68/69) of CaP glands compared with BPE. In 79% of cases (11/14; P < 0.05), mRNA expression was reduced in CaP as compared with patient-matched BPE. Annexin I mRNA and protein expression levels did not correlate with Gleason grade, pathological stage, or race. CONCLUSIONS: Down-regulation of annexin I protein expression is a common finding in HGPIN and CaP, suggesting that annexin I dysregulation may be an important early event in CaP initiation. Because mRNA levels are reduced in a high proportion of cases, one likely mechanism for annexin I dysregulation occurs at the level of gene transcription. Results of these studies support a valuable role for a molecular profiling approach to CaP research.