RESUMEN
OBJECTIVES: To evaluate the relationship between stathmin expression and clinical outcome in colorectal cancer (CRC). BACKGROUND: Stathmin is a phosphoprotein involved in the regulation of microtubule dynamics and integration of intracellular signaling pathways. Stathmin has been implicated in the tumorigenesis of several cancers and is a potential therapeutic target. METHODS: Stathmin expression was evaluated in 25 metastatic CRC (mCRC) patients by immunohistochemistry (IHC). Ki67 IHC and TUNEL assay were also evaluated in mCRC for cell proliferation and apoptosis. RESULTS: High expression of stathmin was correlated with CRC metastasis (p = .0084), and significantly worse overall survival (OS) in CRC patients (p = .036). There was a significant increase in cell proliferation and a decrease in apoptosis in liver metastasis compared with CRC primary tumors as determined by Ki67 IHC and TUNEL assay (p < .0001). We also observed a significant positive correlation between stathmin level and cell proliferation in both CRC primary tumor and liver metastasis (p = .0429 to 0.0451; r = .4236 to .4288). CONCLUSION: Stathmin expression correlated with worse patient prognosis in mCRC patients and positively correlated with increased cell proliferation. Together, our findings indicate stathmin as a novel potential marker for increased risk of CRC-specific mortality and identify stathmin as an attractive therapeutic target for the treatment of mCRC.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Estatmina/metabolismo , Adenocarcinoma/mortalidad , Anciano , Neoplasias Colorrectales/mortalidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Tasa de SupervivenciaRESUMEN
OBJECTIVES: To evaluate the relationship between phosphatase of regenerating liver 3 (PRL3) expression and clinical outcome in colorectal cancer (CRC). BACKGROUND: PRL3, a protein tyrosine phosphatase functions as one of the key regulatory enzymes of various signal transduction pathways. PRL3 is highly expressed in a majority of cancers and is a novel potential therapeutic target. METHODS: PRL3 expression was evaluated by immunohistochemistry in 167 patients with CRC, 37 patients with no disease, and 26 patients with metastatic CRC (mCRC). Phosphorylated Akt at serine 473 (p-Akt S473) expression was also evaluated by immunohistochemistry in mCRC patients. RESULTS: High expression of PRL3 was correlated with CRC progression, and every one unit increase in PRL3 level contributed to an increase in the rate of death by 1%-1.7%. PRL3 expression was significantly higher in liver metastases compared with primary tumors and showed a significant positive correlation with the expression level of p-Akt S473. CONCLUSION: PRL3 expression levels associated with CRC progression and metastasis, and positively correlated with activated Akt level in mCRC. Together, these findings indicated that PRL3 might be a potential marker for increased risk of CRC-specific tumor burden and identify PRL3 as an attractive therapeutic target for mCRC treatment.
Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Carcinoma de Células en Anillo de Sello/patología , Neoplasias Colorrectales/patología , Proteínas de Neoplasias/metabolismo , Recurrencia Local de Neoplasia/patología , Proteínas Tirosina Fosfatasas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Anciano , Carcinoma de Células en Anillo de Sello/metabolismo , Carcinoma de Células en Anillo de Sello/terapia , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/terapia , Terapia Combinada , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/terapia , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Na+ /H+ exchanger regulatory factor 1 (NHERF1) has been implicated in the tumorigenesis of several cancer types and is a potential therapeutic target. The current study evaluated the relationship between NHERF1 expression and clinical outcome in colorectal cancer (CRC). METHODS: NHERF1 expression was evaluated by immunohistochemistry in 167 patients with CRC primary tumors, 37 patients with no disease, and 27 patients with metastatic CRC (mCRC); and in the orthotopically implanted tumors in mice. NHERF1 expression was manipulated in CRC cells using inducible short hairpin RNAs to determine its biological functions. RESULTS: High expression of NHERF1 correlated with CRC progression and metastasis, as well as significantly worse overall survival, recurrence-free survival, and disease-specific survival. Orthotopic implantation studies demonstrated increased NHERF1 expression in liver metastases. Treatment of CRC xenografts with insulin-like growth factor 1 receptor (IGF1R) inhibitors downregulated NHERF1 expression, indicating NHERF1 is downstream of IGF1R signaling. Knockdown of NHERF1 increased apoptosis and reduced X-linked inhibitor of apoptosis protein (XIAP) and survivin expression, indicating NHERF1 is critical for CRC cell survival. CONCLUSION: NHERF1 expression levels correlated with worse prognosis in patients with CRC and plays a critical role in CRC cell survival. Together, our findings establish NHERF1 as a novel potential marker for increased risk of CRC-specific mortality and identify NHERF1 as an attractive therapeutic target for mCRC treatment.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Fosfoproteínas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Anciano , Animales , Biomarcadores de Tumor/biosíntesis , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Células HCT116 , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Tasa de SupervivenciaRESUMEN
Aberrant cell survival plays a critical role in cancer progression and metastasis. We have previously shown that ezrin, a cAMP-dependent protein kinase A-anchoring protein (AKAP), is up-regulated in colorectal cancer (CRC) liver metastasis. Phosphorylation of ezrin at Thr-567 activates ezrin and plays an important role in CRC cell survival associated with XIAP and survivin up-regulation. In this study, we demonstrate that in FET and GEO colon cancer cells, knockdown of ezrin expression or inhibition of ezrin phosphorylation at Thr-567 increases apoptosis through protein kinase A (PKA) activation in a cAMP-independent manner. Transforming growth factor (TGF) ß signaling inhibits ezrin phosphorylation in a Smad3-dependent and Smad2-independent manner and regulates pro-apoptotic function through ezrin-mediated PKA activation. On the other hand, ezrin phosphorylation at Thr-567 by insulin-like growth factor 1 receptor (IGF1R) signaling leads to cAMP-dependent PKA activation and enhances cell survival. Further studies indicate that phosphorylated ezrin forms a complex with PKA RII, and dephosphorylated ezrin dissociates from the complex and facilitates the association of PKA RII with AKAP149, both of which activate PKA yet lead to either cell survival or apoptosis. Thus, our studies reveal a novel mechanism of differential PKA activation mediated by TGFß and IGF1R signaling through regulation of ezrin phosphorylation in CRC, resulting in different cell fates. This is of significance because TGFß and IGF1R signaling pathways are well-characterized tumor suppressor and oncogenic pathways, respectively, with important roles in CRC tumorigenesis and metastasis. Our studies indicate that they cross-talk and antagonize each other's function through regulation of ezrin activation. Therefore, ezrin may be a potential therapeutic target in CRC.
Asunto(s)
Apoptosis , Neoplasias del Colon/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores de Somatomedina/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas del Citoesqueleto/genética , Humanos , Fosforilación , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Transducción de Señal , Células Tumorales CultivadasRESUMEN
BACKGROUND: There is extensive evidence for the role of aberrant cell survival signaling mechanisms in cancer progression and metastasis. Akt is a major component of cell survival-signaling mechanisms in several types of cancer. It has been shown that activated Akt stabilizes XIAP by S87 phosphorylation leading to survivin/XIAP complex formation, caspase inhibition and cytoprotection of cancer cells. We have reported that TGFß/PKA/PP2A-mediated tumor suppressor signaling regulates Akt phosphorylation in association with the dissociation of survivin/XIAP complexes leading to inhibition of stress-dependent induction of cell survival. METHODS: IGF1R-dependent colon cancer cells (GEO and CBS) were used for the study. Effects on cell proliferation and cell death were determined in the presence of MK-2206. Xenograft studies were performed to determine the effect of MK-2206 on tumor volume. The effect on various cell death markers such as XIAP, survivin, AIF, Ezrin, pEzrin was determined by western blot analysis. Graph pad 5.0 was used for statistical analysis. P < 0.05 was considered significant. RESULTS: We characterized the mechanisms by which a novel Akt kinase inhibitor MK-2206 induced cell death in IGF1R-dependent colorectal cancer (CRC) cells with upregulated PI3K/Akt signaling in response to IGF1R activation. MK-2206 treatment generated a significant reduction in tumor growth in vivo and promoted cell death through two mechanisms. This is the first report demonstrating that Akt inactivation by MK-2206 leads to induction of and mitochondria-to-nuclear localization of the Apoptosis Inducing Factor (AIF), which is involved in caspase-independent cell death. We also observed that exposure to MK-2206 dephosphorylated Ezrin at the T567 site leading to the disruption of Akt-pEzrin-XIAP cell survival signaling. Ezrin phosphorylation at this site has been associated with malignant progression in solid tumors. CONCLUSION: The identification of these 2 novel mechanisms leading to induction of cell death indicates MK-2206 might be a potential clinical candidate for therapeutic targeting of the subset of IGF1R-dependent cancers in CRC.
Asunto(s)
Antineoplásicos/farmacología , Factor Inductor de la Apoptosis/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Animales , Apoptosis/efectos de los fármacos , Factor Inductor de la Apoptosis/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Mutational loss of tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) is associated with malignant progression in many cancers, including colorectal cancer (CRC). PTEN is involved in negatively regulating the phosphatidylinositol 3-kinase/AKT oncogenic signaling pathway and has been implicated in the metastatic colonization process. Few in vivo models are available to study CRC metastasis. The purpose of this study was to determine the effect of restoring PTEN activity on metastases in an orthotopic murine model. METHODS: Green fluorescent protein labeled TENN, a highly metastatic human colon cancer cell line with mutational loss of PTEN gene and TENN clones (with restoration of PTEN gene) tumors were orthotopically implanted onto the colons of BALB/c nude mice and allowed to develop primary and metastatic tumors. Seven weeks post-implantation, mice were euthanized and organs extracted for examination. RESULTS: Both TENN and TENN clone cell lines demonstrated 100% primary invasion. However, compared with the parental TENN cells, which demonstrated 62% metastases to both lungs and liver, TENN clone cells showed an approximately 50% reduction in metastasis, with only 31.6% liver metastasis and no metastasis to the lungs (P = 0.02). CONCLUSIONS: Our study shows that reactivation of PTEN tumor suppressor pathway leads to a 50% reduction in CRC metastasis without affecting primary tumor formation. Importantly, PTEN restoration also changed the organotropic homing from liver and lung metastasis to liver metastasis only. This in vivo study demonstrates that PTEN might act specifically as a metastasis suppressor and, thus, efforts to target the phosphatidylinositol 3-kinase/PTEN pathway are legitimate.
Asunto(s)
Neoplasias Colorrectales/patología , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/secundario , Fosfohidrolasa PTEN/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Incidencia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Mutación/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiologíaRESUMEN
Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor ß receptor II (TGFßRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFß signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFß signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFß/protein kinase A signaling pathway. Induction of TGFßRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFßRII might potentially benefit from the use of this histone deacetylase inhibitor.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Catálisis , Muerte Celular , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Epigénesis Genética , Humanos , ARN Interferente Pequeño/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Sulfonamidas , Survivin , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: TGFß signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFß signaling. METHODS: To test the importance of TGFß signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFß response (FET), or tumorigenic with TGFß response (FETα) or tumorigenic with abrogated TGFß response via introduction of dominant negative TGFßRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging. RESULTS: Abrogation of TGFß signaling through introduction of a dominant negative TGFß receptor II (TGFßRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFß signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFß signaling is a metastasis suppressor, we rescued TGFß signaling in CBS metastatic colon cancer cells that had lost TGFß receptor expression due to epigenetic repression. Restoration of TGFß signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFß signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma. CONCLUSIONS: The observations presented here indicate a metastasis suppressor role for TGFß signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFß signaling may be imprudent in some patient populations with residual TGFß tumor suppressor activity.
Asunto(s)
Neoplasias del Colon/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células HEK293 , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Trasplante Heterólogo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
The alpha isoform of the phosphatidylinositol-3-kinases (PI3Kα) is often mutated, amplified and overexpressed in human tumors. In an effort to develop new inhibitors targeting this enzyme, we carried out a pharmacophore model study based on six PI3Kα-selective compounds. The pharmacophore searching identified three structurally novel inhibitors of PI3Kα and its H1047R mutant. Our biological studies show that two of our hit molecules suppressed the formation of pAKT, a downstream effector of PI3Kα, and induced apoptosis in the HCT116 colon cancer cell line. QPLD-based docking showed that residues Asp933, Glu849, Val851, and Gln859 appeared to be key binding residues for active inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Estructura Molecular , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
This work describes our efforts to optimize the lead PI3Kα inhibitor N-benzyl 4-hydroxy-2-quinolone-3-carboxamide using structure-based design and molecular docking. We identified a series of N-phenyl 4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R versus wild-type PI3Kα and we also showed that the cell growth inhibition by these compounds likely occurs by inhibiting the formation of pAKT and induction of apoptosis.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/química , Quinolinas/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Simulación del Acoplamiento Molecular , Mutación , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.23749.].
RESUMEN
In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin-mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA-MB-231 and PC-3 cells were plated on VN was enhanced. Further experiments showed co-immunoprecipitation of integrins beta1, alpha V beta 3, or alpha V beta 5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss-of-function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrinas/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Neoplasias/metabolismo , Transducción de Señal/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Integrina beta1/metabolismo , Integrinas/genética , Proteína Quinasa 7 Activada por Mitógenos/genética , Neoplasias/patología , Receptores de Vitronectina/metabolismo , Vitronectina/metabolismoRESUMEN
Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Morfolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Procesos de Crecimiento Celular , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador alfa/antagonistas & inhibidores , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in the PIK3CA gene are common in human cancers, including colon cancer. We compared two pairs of colon cancer cells (HCT116 and DLD1) bearing only the wild-type (WT) or mutant (MUT) PIK3CA allele for their survival capacity under stress conditions in vitro as well as their metastatic properties in an in vivo orthotopic model. When subjected to growth factor deprivation stress (GFDS), the MUT PIK3CA cells displayed resistance to GFDS-induced apoptosis relative to the WT cells. Phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT were constitutively activated during stress conditions in the MUT PIK3CA cells but not in the WT cells. The MUT cells showed hypersensitivity to PI3K inhibition. Moreover, the proapoptotic protein Bax was expressed at a very high level in the WT PIK3CA cells, whereas it was almost undetectable in the MUT cells. Inhibition of Bax expression by small interfering RNA protected the WT PIK3CA cells from GFDS-induced apoptosis, suggesting an important role of Bax in GFDS-induced apoptosis. These results indicated that the MUT PI3K confers resistance to GFDS-induced apoptosis and that the MUT cells are more dependent on the PI3K pathway for survival. In vivo studies showed that the MUT PIK3CA-bearing cells were more metastatic than the WT cells in an orthotopic model of colon cancer. Taken together, these results suggest that MUT PI3K imparts a more aggressive phenotype in colon cancer cells and could be a potential therapeutic target for treatment of colon cancer patients bearing PIK3CA mutations.
Asunto(s)
Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Invasividad Neoplásica/fisiopatología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias del Colon/genética , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Desnudos , Mutación , Metástasis de la Neoplasia/fisiopatología , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of <10% due in part to a lack of effective therapies. Pan-histone deacetylase (HDAC) inhibitors have shown preclinical efficacy against PDAC but have failed in the clinic due to toxicity. Selective HDAC inhibitors may reduce toxicity while retaining therapeutic efficacy. However, their use requires identification of the specific HDACs that mediate the therapeutic effects of HDAC inhibitors in PDAC. We determined that the HDAC1/2/3 inhibitor Mocetinostat synergizes with the HDAC4/5/6 inhibitor LMK-235 in a panel of PDAC cell lines. Furthermore, while neither drug alone synergizes with gemcitabine, the combination of Mocetinostat, LMK-235, and gemcitabine showed strong synergy. Using small interfering (si)RNA-mediated knockdown, this synergy was attributed to inhibition of HDACs 1, 2, and 6. Pharmacological inhibition of HDACs 1 and 2 with Romidepsin and HDAC6 with ACY-1215 also potently synergized with gemcitabine in a panel of PDAC cell lines, and this drug combination potentiated the antitumor effects of gemcitabine against PDAC xenografts in vivo. Collectively, our data show that inhibition of multiple HDACs is required for therapeutic effects of HDAC inhibitors and support the development of novel strategies to inhibit HDACs 1, 2, and 6 for PDAC therapy.
RESUMEN
PURPOSE: In preclinical models, calcitriol and the tyrosine kinase inhibitor gefitinib are synergistic and modulate extracellular signal-regulated kinase (Erk) and Akt pathways. Therefore, we conducted a phase I study of calcitriol and gefitinib to determine the maximum tolerated dose (MTD) of this combination. EXPERIMENTAL DESIGN: Calcitriol was given i.v. over 1 h on weeks 1, 3, and weekly thereafter. Gefitinib was given at a fixed oral daily dose of 250 mg starting at week 2 (day 8). Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for calcitriol and gefitinib. Serial skin biopsies were done to investigate epidermal growth factor receptor (EGFR) pathway pharmacodynamic interactions. RESULTS: Thirty-two patients were treated. Dose-limiting hypercalcemia was noted in two of four patients receiving 96 mug/wk of calcitriol. One of seven patients developed dose-limiting hypercalcemia at the MTD 74 mug/wk calcitriol dose level. The relationship between calcitriol dose and peak serum calcitriol (C(max)) and systemic exposure (AUC) was linear. Mean (+/-SD) serum calcitriol C(max) at the MTD was 6.68 +/- 1.42 ng/mL. Gefitinib treatment inhibited EGFR, Akt, and Erk phosphorylation in the skin. Calcitriol did not have consistent effects on skin EGFR or its downstream elements. The combination of gefitinib and calcitriol did not modulate tumor EGFR pathway in patients with serial tumor biopsies. CONCLUSIONS: High doses of weekly i.v. calcitriol can be administered safely in combination with gefitinib. Calcitriol concentrations achieved at the MTD 74 mug calcitriol exceed in vivo concentrations associated with antitumor activity in preclinical models.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Calcitriol/efectos adversos , Calcitriol/farmacocinética , Calcitriol/farmacología , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Femenino , Gefitinib , Humanos , Inmunohistoquímica , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Neoplasias/enzimología , Fosforilación , Quinazolinas/efectos adversos , Quinazolinas/farmacocinética , Quinazolinas/farmacologíaRESUMEN
Coexpression of the epidermal growth factor receptor (EGFR) family receptors is found in a subset of colon cancers, which may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. Recently, efforts have been made to develop novel 4-anilinoquinazoline and pyridopyrimidine derivatives to inhibit EGFR and ErbB2 kinases simultaneously. In this study, we tested the efficacy of a novel reversible dual inhibitor GW572016 compared with the selective EGFR and ErbB2 tyrosine kinase inhibitors (TKI) AG1478 and AG879 and their combination, using the human colon adenocarcinoma GEO mode. GEO cells depend on multiple ErbB receptors for aberrant growth. A synergistic effect on inhibition of cell proliferation associated with induction of apoptosis was observed from the combination of AG1478 and AG879. Compared with AG1478 or AG879, the single TKI compound GW572016 was a more potent inhibitor of GEO cell proliferation and was able to induce apoptosis at lower concentrations. Western blot analysis revealed that AG1478 and AG879 were unable to suppress both EGFR and ErbB2 activation as well as the downstream mitogen-activated protein kinase (MAPK) and AKT pathways as single agents. In contrast, GW572016 suppressed the activation of EGFR, ErbB2, MAPK, and AKT in a concentration-dependent manner. Finally, in vivo studies showed that GW572016 treatment efficiently blocked GEO xenograft growth at a dose range of 30 to 200 mg/kg with a twice-daily schedule. In summary, our study indicates that targeting both EGFR and ErbB2 simultaneously could enhance therapy over that of single agents directed at EGFR or ErbB2 in cancers that can be identified as being primarily heterodimer-dependent.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Humanos , Lapatinib , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Tirfostinos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias del Colon/genética , Resistencia a Antineoplásicos , Sustancias de Crecimiento/deficiencia , Mutación/genética , Fosfatidilinositol 3-Quinasas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 - now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was â¼3.5-fold and â¼65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average >2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy.
RESUMEN
It has generally been believed that the diffusion limit set by the nuclear pore for protein is 60kDa. We here studied the cellular localization of several artificial proteins and found that the diffusion limit set by the nuclear pore is not as small as previously thought. The results indicate that the maximal size of protein to diffuse through the nuclear pore complex could be quite larger than 60kDa, thus greatly extending the diffusion limit that the nuclear pore can accommodate.