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1.
Proc Natl Acad Sci U S A ; 109(29): 11717-22, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22753465

RESUMO

The Wnt/ß-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Receptores Frizzled/metabolismo , Neoplasias/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Sinergismo Farmacológico , Vetores Genéticos/genética , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imuno-Histoquímica , Lentivirus , Luciferases , Neoplasias/metabolismo , Biblioteca de Peptídeos , Via de Sinalização Wnt/fisiologia
2.
N Engl J Med ; 356(3): 217-26, 2007 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-17229949

RESUMO

BACKGROUND: Breast cancers contain a minority population of cancer cells characterized by CD44 expression but low or undetectable levels of CD24 (CD44+CD24-/low) that have higher tumorigenic capacity than other subtypes of cancer cells. METHODS: We compared the gene-expression profile of CD44+CD24-/low tumorigenic breast-cancer cells with that of normal breast epithelium. Differentially expressed genes were used to generate a 186-gene "invasiveness" gene signature (IGS), which was evaluated for its association with overall survival and metastasis-free survival in patients with breast cancer or other types of cancer. RESULTS: There was a significant association between the IGS and both overall and metastasis-free survival (P<0.001, for both) in patients with breast cancer, which was independent of established clinical and pathological variables. When combined with the prognostic criteria of the National Institutes of Health, the IGS was used to stratify patients with high-risk early breast cancer into prognostic categories (good or poor); among patients with a good prognosis, the 10-year rate of metastasis-free survival was 81%, and among those with a poor prognosis, it was 57%. The IGS was also associated with the prognosis in medulloblastoma (P=0.004), lung cancer (P=0.03), and prostate cancer (P=0.01). The prognostic power of the IGS was increased when combined with the wound-response (WR) signature. CONCLUSIONS: The IGS is strongly associated with metastasis-free survival and overall survival for four different types of tumors. This genetic signature of tumorigenic breast-cancer cells was even more strongly associated with clinical outcomes when combined with the WR signature in breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Perfilação da Expressão Gênica , Mama/patologia , Neoplasias da Mama/patologia , Antígeno CD24 , Epitélio , Feminino , Humanos , Receptores de Hialuronatos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Meduloblastoma/genética , Meduloblastoma/mortalidade , Invasividade Neoplásica , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade
3.
PLoS One ; 15(3): e0229445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160239

RESUMO

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.


Assuntos
Anticorpos/uso terapêutico , Fibrose Pulmonar Idiopática , Hepatopatia Gordurosa não Alcoólica , Trombospondinas/fisiologia , Animais , Células Cultivadas , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
4.
Stem Cells ; 26(2): 364-71, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17975224

RESUMO

In human breast cancers, a phenotypically distinct minority population of tumorigenic (TG) cancer cells (sometimes referred to as cancer stem cells) drives tumor growth when transplanted into immunodeficient mice. Our objective was to identify a mouse model of breast cancer stem cells that could have relevance to the study of human breast cancer. To do so, we used breast tumors of the mouse mammary tumor virus (MMTV)-Wnt-1 mice. MMTV-Wnt-1 breast tumors were harvested, dissociated into single-cell suspensions, and sorted by flow cytometry on Thy1, CD24, and CD45. Sorted cells were then injected into recipient background FVB/NJ female syngeneic mice. In six of seven tumors examined, Thy1+CD24+ cancer cells, which constituted approximately 1%-4% of tumor cells, were highly enriched for cells capable of regenerating new tumors compared with cells of the tumor that did not fit this profile ("not-Thy1+CD24+"). Resultant tumors had a phenotypic diversity similar to that of the original tumor and behaved in a similar manner when passaged. Microarray analysis comparing Thy1+CD24+ tumor cells to not-Thy1+CD24+ cells identified a list of differentially expressed genes. Orthologs of these differentially expressed genes predicted survival of human breast cancer patients from two different study groups. These studies suggest that there is a cancer stem cell compartment in the MMTV-Wnt-1 murine breast tumor and that there is a clinical utility of this model for the study of cancer stem cells.


Assuntos
Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Separação Celular/métodos , Feminino , Citometria de Fluxo , Humanos , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Células-Tronco Neoplásicas/classificação , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Retroviridae/genética , Infecções por Retroviridae/patologia , Transplante Isogênico , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologia , Proteína Wnt1/genética
5.
Cancer Res ; 79(5): 970-981, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30622113

RESUMO

A subset of patients with gastric cancer have mutations in genes that participate in or regulate Wnt signaling at the level of ligand (Wnt) receptor (Fzd) binding. Moreover, increased Fzd expression is associated with poor clinical outcome. Despite these findings, there are no in vivo studies investigating the potential of targeting Wnt receptors for treating gastric cancer, and the specific Wnt receptor transmitting oncogenic Wnt signaling in gastric cancer is unknown. Here, we use inhibitors of Wnt/Fzd (OMP-18R5/vantictumab) and conditional gene deletion to test the therapeutic potential of targeting Wnt signaling in preclinical models of intestinal-type gastric cancer and ex vivo organoid cultures. Pharmacologic targeting of Fzd inhibited the growth of gastric adenomas in vivo. We identified Fzd7 to be the predominant Wnt receptor responsible for transmitting Wnt signaling in human gastric cancer cells and mouse models of gastric cancer, whereby Fzd7-deficient cells were retained in gastric adenomas but were unable to respond to Wnt signals and consequently failed to proliferate. Genetic deletion of Fzd7 or treatment with vantictumab was sufficient to inhibit the growth of gastric adenomas with or without mutations to Apc. Vantictumab is currently in phase Ib clinical trials for advanced pancreatic, lung, and breast cancer. Our data extend the scope of patients that may benefit from this therapeutic approach as we demonstrate that this drug will be effective in treating patients with gastric cancer regardless of APC mutation status. SIGNIFICANCE: The Wnt receptor Fzd7 plays an essential role in gastric tumorigenesis irrespective of Apc mutation status, therefore targeting Wnt/Fzd7 may be of therapeutic benefit to patients with gastric cancer.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Receptores Frizzled/metabolismo , Neoplasias Gástricas/metabolismo , Via de Sinalização Wnt , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Carcinogênese , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Receptores Frizzled/antagonistas & inibidores , Receptores Frizzled/genética , Deleção de Genes , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Mutação , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
6.
Nat Commun ; 10(1): 5016, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676788

RESUMO

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1ß stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1ß-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.


Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Interleucina-1beta/metabolismo , Células-Tronco Neoplásicas/metabolismo , Via de Sinalização Wnt , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Sulfassalazina/administração & dosagem , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Neurosci ; 6(12): 1270-6, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14595443

RESUMO

Netrin-G1 is a lipid-anchored protein that is structurally related to the netrin family of axon guidance molecules. Netrin-G1 does not bind any of the known netrin receptors and its function is not known. Here we identify human netrin-G1 ligand (NGL-1), a transmembrane protein containing leucine-rich repeat (LRR) and immunoglobulin (Ig) domains that specifically interacts with netrin-G1 through its LRR region. Whereas netrin-G1 is expressed highly in mouse thalamic axons, NGL-1 is most abundant in the striatum and the cerebral cortex--the intermediate and final targets, respectively, of thalamocortical axons (TCAs). Surface-bound NGL-1 stimulates, but soluble NGL-1 disrupts, the growth of embryonic thalamic axons, and in vitro data indicate that NGL-1 activity may be mediated at least partially by netrin-G1. Our findings provide evidence that netrin-G1 functions as an important component of the NGL-1 receptor to promote TCA outgrowth and that membrane-bound netrins can participate in receiving axonal signaling pathways.


Assuntos
Axônios/metabolismo , Córtex Cerebral/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/fisiologia , Tálamo/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Córtex Cerebral/embriologia , Embrião de Galinha , Feminino , Humanos , Ligantes , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Receptores de Netrina , Netrinas , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Superfície Celular/metabolismo , Tálamo/embriologia
8.
Sci Adv ; 3(6): e1700090, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28691093

RESUMO

The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.


Assuntos
Antineoplásicos/farmacologia , Mitose/efeitos dos fármacos , Taxoides/farmacologia , Proteínas Wnt/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Paclitaxel/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores
9.
Cancer Res ; 76(3): 713-23, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26719531

RESUMO

Deregulation of the ß-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and ß-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate ß-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate ß-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited ß-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of ß-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.


Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Trombospondinas/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Trombospondinas/biossíntese , Trombospondinas/genética , Trombospondinas/imunologia , Proteínas Wnt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Leukoc Biol ; 71(1): 1-8, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11781375

RESUMO

IL-17 is a potent proinflammatory cytokine produced by activated memory T cells. The large-scale sequencing of the human and other vertebrate genomes has revealed the presence of additional genes encoding proteins clearly related to IL-17, thus defining a new family of cytokines. There are at least six members of the IL-17 family in humans and in mice. Initial characterization suggests that like IL-17, several of these newly identified molecules have the ability to modulate immune function. Neither the IL-17 family nor the cognate receptors that have been identified for these molecules bear obvious sequence similarity to other known families of proteins. Thus, they represent a distinct signaling system that appears to have been highly conserved across vertebrate evolution. The potent inflammatory actions that have been identified for several of these factors and the emerging associations with major human diseases suggest that these proteins may have significant roles in inflammatory processes.


Assuntos
Interleucina-17/imunologia , Transdução de Sinais/imunologia , Animais , Evolução Molecular , Humanos , Interleucina-17/genética , Camundongos , Linfócitos T/imunologia
11.
Clin Cancer Res ; 21(9): 2084-95, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25934888

RESUMO

PURPOSE: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. EXPERIMENTAL DESIGN: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. CONCLUSIONS: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor Notch2/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Animais , Humanos , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch3 , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Int Immunopharmacol ; 4(5): 669-77, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15120651

RESUMO

IL-22, also termed IL-TIF, is a member of the IL-10 family of cytokines. Its principal source appears to be memory CD4 T cells with a Th1 polarized phenotype. IL-22 induces its signals through a two-component receptor comprised of IL-22R1 and CRF2-4/IL10Rb. Both of these receptor components also participate in separate receptor complexes specific for other IL-10 family cytokines. Because CRF2-4 exhibits ubiquitous expression, the tropism of IL-22 action appears to be dictated by the expression of IL-22R1. IL-22R1 has a highly restricted expression pattern. Its highest expression, by far, is in the acinar cell population of the pancreas. Lower, but still functional, levels of expression are also observed in skin, colon, liver, and kidney. The responses that have been observed to date for IL-22 resemble the "acute phase" type responses elicited by IL-6, suggesting that IL-22 might be appropriately considered as a T cell-derived IL-6-like activity having distinct target cell specificity. The functional role of this system remains unclear, but it is likely that the responses elicited by this cytokine serve to contribute both to acute host defense against pathogens and to safeguard vulnerable target tissues under conditions of stress.


Assuntos
Citocinas/imunologia , Interleucinas/imunologia , Especificidade de Órgãos/efeitos dos fármacos , Pâncreas/citologia , Células Th1/imunologia , Citocinas/metabolismo , Citocinas/farmacologia , Humanos , Interleucinas/genética , Interleucinas/farmacologia , Especificidade de Órgãos/imunologia , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th1/metabolismo , Interleucina 22
13.
Cancer Res ; 73(15): 4909-22, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23761328

RESUMO

Wnt ligand expression and activation of the Wnt/ß-catenin pathway have been associated with pancreatic ductal adenocarcinoma, but whether Wnt activity is required for the development of pancreatic cancer has remained unclear. Here, we report the results of three different approaches to inhibit the Wnt/ß-catenin pathway in a established transgenic mouse model of pancreatic cancer. First, we found that ß-catenin null cells were incapable of undergoing acinar to ductal metaplasia, a process associated with development of premalignant pancreatic intraepithelial neoplasia lesions. Second, we addressed the specific role of ligand-mediated Wnt signaling through inducible expression of Dkk1, an endogenous secreted inhibitor of the canonical Wnt pathway. Finally, we targeted the Wnt pathway with OMP-18R5, a therapeutic antibody that interacts with multiple Frizzled receptors. Together, these approaches showed that ligand-mediated activation of the Wnt/ß-catenin pathway is required to initiate pancreatic cancer. Moreover, they establish that Wnt signaling is also critical for progression of pancreatic cancer, a finding with potential therapeutic implications.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Pancreáticas/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Apoptose/fisiologia , Western Blotting , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , beta Catenina/metabolismo
14.
Clin Cancer Res ; 18(19): 5374-86, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22952347

RESUMO

PURPOSE: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy. EXPERIMENTAL DESIGN: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays. RESULTS: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition. CONCLUSIONS: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Neovascularização Patológica/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptores Notch/imunologia , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Gencitabina
15.
Vasc Cell ; 3: 18, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21831306

RESUMO

DLL4 is a ligand for the Notch family of receptors. DLL4 has many important functions in normal development and tissue homeostasis, including roles in the immune system, the gastro-intestinal tract, and in vascular development. Because of the importance of Notch signaling in stem cell biology, DLL4 has been investigated for its role in the maintenance and proliferation of cancer stem cells (CSC). In addition, its important role in angiogenesis has been investigated for utility as an anti-angiogenic agent. Preclinical studies have highlighted that both anti-CSC and anti-angiogenic activities contribute to its anti-tumor efficacy, and have supported the clinical development of anti-DLL4 antibody for the treatment of cancer.

16.
Cancer Res ; 71(5): 1520-5, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21193546

RESUMO

KRAS mutations are frequent in colorectal cancer (CRC) and are associated with clinical resistance to treatment with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway and mediates stem cell self-renewal and vascular development. DLL4 inhibition in colon tumor cells reduces tumor growth and stem cell frequency. Considering the need for new drugs to treat colon cancers with oncogenic KRAS mutations, we examined in this study the efficacy of anti-DLL4 antibodies in KRAS mutant tumors in a panel of early passage colon tumor xenograft models derived from patients. Consistent with clinical findings, mutant KRAS colorectal xenograft tumors were insensitive to the EGFR therapeutic antibody cetuximab, whereas KRAS wild-type tumors responded to cetuximab. In contrast, anti-DLL4 was efficacious against both wild-type and mutant KRAS colon tumors as a single agent and in combination with irinotecan. Further analysis of mutant KRAS tumors indicated that the anti-DLL4/irinotecan combination produced a significant decrease in colon cancer stem cell frequency while promoting apoptosis in tumor cells. Our findings provide a rationale for targeting DLL4-Notch signaling for improved treatment of CRC patients with activating KRAS mutations.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Irinotecano , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto
17.
PLoS One ; 4(9): e7002, 2009 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-19759823

RESUMO

BACKGROUND: Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. PRINCIPAL FINDINGS: To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. CONCLUSION: The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Técnicas Genéticas , Códon , DNA/genética , Proteínas de Escherichia coli/genética , Genes Sintéticos , Engenharia Genética , Análise dos Mínimos Quadrados , Modelos Genéticos , Fases de Leitura Aberta , Engenharia de Proteínas/métodos , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
18.
Cell Stem Cell ; 5(2): 168-77, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19664991

RESUMO

Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Neoplasias/terapia , Células-Tronco Neoplásicas/imunologia , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chaperonina 60/agonistas , Chaperonina 60/metabolismo , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Irinotecano , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neovascularização Patológica/metabolismo , Prevenção Secundária , Ensaios Antitumorais Modelo de Xenoenxerto
19.
PLoS One ; 3(6): e2428, 2008 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-18560594

RESUMO

BACKGROUND: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. METHODS AND FINDINGS: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. CONCLUSIONS: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Ciclofosfamida/uso terapêutico , Células-Tronco Neoplásicas/citologia , Aldeído Desidrogenase/genética , Animais , Camptotecina/uso terapêutico , Humanos , Irinotecano , Camundongos
20.
Proc Natl Acad Sci U S A ; 104(24): 10158-63, 2007 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-17548814

RESUMO

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the "cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM)(high)/CD44+ epithelial cells. Tumors originated from EpCAM(high)/CD44+ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM(high)/CD44+ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.


Assuntos
Antígenos de Neoplasias , Biomarcadores Tumorais/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Células-Tronco/imunologia , Molécula de Adesão de Leucócito Ativado/imunologia , Animais , Antígenos de Neoplasias/imunologia , Separação Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Células Epiteliais/imunologia , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fenótipo , Transplante Heterólogo , Ensaio Tumoral de Célula-Tronco
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