RESUMO
Occurrence of resistance to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) approved in ovarian carcinoma, has already been shown in clinical settings. Identifying combination treatments to sensitize tumor cells and/or overcome resistance to olaparib is critical. Polo-like kinase 1 (PLK1), a master regulator of mitosis, is also involved in the DNA damage response promoting homologous recombination (HR)-mediated DNA repair and in the recovery from the G2/M checkpoint. We hypothesized that PLK1 inhibition could sensitize tumor cells to PARP inhibition. Onvansertib, a highly selective PLK1 inhibitor, and olaparib were tested in vitro and in vivo in BRCA1 mutated and wild-type (wt) ovarian cancer models, including patient-derived xenografts (PDXs) resistant to olaparib. The combination of onvansertib and olaparib was additive or synergic in different ovarian cancer cell lines, causing a G2/M block of the cell cycle, DNA damage, and apoptosis, much more pronounced in cells treated with the two drugs as compared to controls and single agents treated cells. The combined treatment was well tolerated in vivo and resulted in tumor growth inhibition and a statistically increased survival in olaparib-resistant-BRCA1 mutated models. The combination was also active, although to a lesser extent, in BRCA1 wt PDXs. Pharmacodynamic analyses showed an increase in mitotic, apoptotic, and DNA damage markers in tumor samples derived from mice treated with the combination versus vehicle. We could demonstrate that in vitro onvansertib inhibited both HR and non-homologous end-joining repair pathways and in vivo induced a decrease in the number of RAD51 foci-positive tumor cells, supporting its ability to induce HR deficiency and favoring the activity of olaparib. Considering that the combination was well tolerated, these data support and foster the clinical evaluation of onvansertib with PARPis in ovarian cancer, particularly in the PARPis-resistant setting.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Ftalazinas , Piperazinas , Inibidores de Poli(ADP-Ribose) Polimerases , Feminino , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Linhagem Celular Tumoral , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Dano ao DNA/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacosRESUMO
We describe the development and validation of an HPLC-MS/MS method to assess the pharmacokinetics and tumour distribution of ZST316, an arginine analogue with inhibitory activity towards dimethylarginine dimethylaminohydrolase 1 (DDAH1) and vasculogenic mimicry, and its active metabolite L-257 in a xenograft model of triple-negative breast cancer (TNBC). The method proved to be reproducible, precise, and highly accurate for the measurement of both compounds in plasma and tumour tissue following acute and chronic (five days) intraperitoneal administration of ZST316 (30 mg/Kg daily) in six-week-old severe combined immunodeficiency disease (SCID) mice inoculated with MDA-MB-231 TNBC cells. ZST316 was detected in tumour tissue and plasma after 1 h (6.47 and 9.01 µM, respectively) and 24 h (0.13 and 0.16 µM, respectively) following acute administration, without accumulation during chronic treatment. Similarly, the metabolite L-257 was found in tumour tissue and plasma after 1 h (15.06 and 8.72 µM, respectively) and 24 h (0.17 and 0.17 µM, respectively) following acute administration of ZST316, without accumulation during chronic treatment. The half-life after acute and chronic treatment ranged between 4.4-7.1 h (plasma) and 4.5-5.0 h (tumour) for ZST316, and 4.2-5.3 h (plasma) and 3.6-4.9 h (tumour) for L-257. The results of our study demonstrate the (a) capacity to accurately measure ZST316 and L-257 concentrations in plasma and tumour tissue in mice using the newly developed HPLC-MS/MS method, (b) rapid conversion of ZST316 into L-257, (c) good intra-tumour penetration of both compounds, and (d) lack of accumulation of both ZST316 and L-257 in plasma and tumour tissue during chronic administration. Compared to a previous method developed by our group to investigate ZST316 in plasma, the main advantages of the new method include a wider range of linearity which reduces the need for dilutions and the combined assessment of ZST316 and L-257 in plasma and tumour tissue which limits the required amount of matrix. The new HPLC-MS/MS method is useful to investigate the in vivo effects of ZST316 and L-257 on vasculogenic mimicry, tumour mass, and metastatic burden in xenograft models of TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Camundongos , Animais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Espectrometria de Massas em Tandem , Xenoenxertos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Chemotherapy is the standard of care for most malignancies. Its tumor debulking effect in adjuvant or neoadjuvant settings is unquestionable, although secondary effects have been reported that paradoxically promote metastasis. Chemotherapy affects the hematopoietic precursors leading to myelosuppression, with neutropenia being the main hematological toxicity induced by cytotoxic therapy. We used renal and lung murine tumor models metastatic to the lung to study chemotherapy-induced neutropenia (CIN) in the metastatic process. Cyclophosphamide and doxorubicin, two myelosuppressive drugs, but not cisplatin, increased the burden of artificial metastases to the lung, by reducing neutrophils. This effect was recapitulated by treatment with anti-Ly6G, the selective antibody-mediated neutrophil depletion that unleashed the formation of lung metastases in both artificial and spontaneous metastasis settings. The increased cancer dissemination was reversed by granulocyte-colony stimulating factor-mediated boosting of neutrophils in combination with chemotherapy. CIN affected the early metastatic colonization of the lung, quite likely promoting the proliferation of tumor cells extravasated into the lung at 24-72 hours. CIN did not affect the late events of the metastatic process, with established metastasis to the lung, nor was there any effect on the release of cancer cells from the primary, whose growth was, in fact, somewhat inhibited. This work suggests a role of neutrophils associated to a common cancer treatment side effect and claims a deep dive into the relationship between chemotherapy-induced neutropenia and metastasis.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Neutropenia , Camundongos , Animais , Neutropenia/induzido quimicamente , Neutropenia/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos/efeitos adversos , Proliferação de CélulasRESUMO
PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair-deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents. We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer. In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors. The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment. These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents. Significance: Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX-bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.
Assuntos
Adenocarcinoma , Neoplasias Ovarianas , Feminino , Humanos , Animais , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Carboplatina/uso terapêutico , Xenoenxertos , Platina/uso terapêutico , Proteína BRCA2 , Neoplasias Ovarianas/tratamento farmacológico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Modelos Animais de Doenças , Adenocarcinoma/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/uso terapêuticoRESUMO
Different tissues have different endothelial features, however, the implications of this heterogeneity in pathological responses are not clear yet. "Inflamm-aging" has been hypothesized as a possible trigger of diseases, including osteoarthritis (OA) and sarcopenia, often present in the same patient. To highlight a possible contribution of organ-specific endothelial cells (ECs), we compare ECs derived from bone and skeletal muscle of the same OA patients. OA bone ECs show a pro-inflammatory signature and higher angiogenic sprouting as compared to muscle ECs, in control conditions and stimulated with TNFα. Furthermore, growth of muscle but not bone ECs decreases with increasing patient age and systemic inflammation. Overall, our data demonstrate that inflammatory conditions in OA patients differently affect bone and muscle ECs, suggesting that inflammatory processes increase angiogenesis in subchondral bone while associated systemic low-grade inflammation impairs angiogenesis in muscle, possibly highlighting a vascular trigger linking OA and sarcopenia.
Assuntos
Células Endoteliais , Sarcopenia , Humanos , Envelhecimento , Músculo Esquelético , Inflamação , EndotélioRESUMO
Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1ß. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1ß (PGC1α/ß) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/ß-expressing OC-PDX-bearing mice. Conversely, low PGC1α/ß OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/ß expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and ß as biomarkers to refine the selection of patients likely to benefit most from this therapy. SIGNIFICANCE: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/ß expression and offers an effective approach to manage patients on the basis of PGC1α/ß expression.
Assuntos
Neoplasias Ovarianas , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA , Animais , Feminino , Humanos , Camundongos , Mitocôndrias/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Ovarian cancer is the deadliest gynecologic cancer, with a 5-year survival rate of 30%, when the disease has spread throughout the peritoneal cavity. We investigated the efficacy to delay disease progression by the DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648, administered in combination with two of the therapeutic options for patient management: either pegylated liposomal doxorubicin (PLD) or the PARP inhibitor olaparib. Patient-derived ovarian cancer xenografts (OC-PDX) were transplanted subcutaneously to evaluate the effect of treatment on tumor growth, or orthotopically in the peritoneal cavity to evaluate the effect on metastatic spread. AZD7648 was administered orally in combination with PLD (dosed intravenously) or with olaparib (orally). To prove the inhibition of DNA-PK in the tumors, we measured pDNA-PKcs, pRPA32, and γH2AX, biomarkers of DNA-PK activity. AZD7648 enhanced the therapeutic efficacy of PLD in all the OC-PDXs tested, regardless of their BRCA status or sensitivity to cisplatin or PLD. The treatment caused disease stabilization, which persisted despite therapy discontinuation for tumors growing subcutaneously, and significantly impaired the abdominal metastatic dissemination, prolonging the lifespan of mice implanted orthotopically. AZD7648 potentiated the efficacy of olaparib in BRCA-deficient OC-PDXs but did not sensitize BRCA-proficient OC-PDXs to olaparib, despite an equivalent inhibition of DNA-PK, suggesting the need of a preexisting olaparib activity to benefit from the addition of AZD7648. This work suggests that AZD7648, an inhibitor of DNA-PK, dosed in combination with PLD or olaparib is an exciting therapeutic option that could benefit patients with ovarian cancer and should be explored in clinical trials.
Assuntos
Neoplasias Ovarianas , Inibidores de Proteínas Quinases , Animais , Linhagem Celular Tumoral , DNA/uso terapêutico , Doxorrubicina/análogos & derivados , Feminino , Xenoenxertos , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Ftalazinas , Piperazinas , Polietilenoglicóis , Inibidores de Proteínas Quinases/uso terapêutico , Purinas , Piranos , TriazóisRESUMO
Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Quinazolinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Genes BRCA1/efeitos dos fármacos , Genes BRCA2/efeitos dos fármacos , Humanos , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.
Assuntos
Processamento Alternativo , Proteínas Angiogênicas/metabolismo , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Cultivadas , Humanos , Antígeno Neuro-Oncológico VentralRESUMO
Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.
Assuntos
Inibidores da Angiogênese/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Células 3T3 , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Proteases contribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy.
Assuntos
Movimento Celular/genética , Células Endoteliais/metabolismo , Glicoproteínas/genética , Neoplasias/genética , Tripsina/genética , Western Blotting , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/metabolismo , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Proteólise , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsina/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Tyrosine kinase inhibitors, affecting angiogenesis, have shown therapeutic efficacy in renal cell carcinoma (RCC). The increased overall survival is not fully explained by their anti-tumor activity, since these drugs frequently induce disease stabilization rather than regression. RCC patients frequently develop cachectic syndrome. We used the RXF393 human renal carcinoma xenograft that recapitulates the characteristics of the disease, including the growth in the mouse kidney (orthotopic implantation), and the induction of cachexia with subsequent premature death. Sunitinib prevents body weight loss and muscle wasting and significantly improves the survival of RXF393-bearing nude mice. The anti-cachectic effect was not associated to direct anti-tumor activity of the drug. Most relevant is the ability of sunitinib to reverse the cachectic phenotype and rescue the animals from the loss of fat tissue. Body weight loss is prevented also in mice bearing the C26 colon carcinoma, classically reported to induce cachexia in immunocompetent mice. Among the mechanisms, we herein show that sunitinib is able to restrain the overactivation of STAT3 and MuRF-1 pathways, involved in enhanced muscle protein catabolism during cancer cachexia. We suggest that off-target effects of angiogenesis inhibitors targeting STAT3 are worth considering as a therapeutic option for patients who develop cachexia, independently of their anti-tumor activity.
Assuntos
Inibidores da Angiogênese/farmacologia , Caquexia/prevenção & controle , Carcinoma de Células Renais/tratamento farmacológico , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caquexia/diagnóstico por imagem , Caquexia/metabolismo , Caquexia/patologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Sunitinibe , Fatores de Tempo , Proteínas com Motivo Tripartido , Carga Tumoral/efeitos dos fármacos , Redução de Peso/efeitos dos fármacos , Microtomografia por Raio-X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Differently from most transformed cells, cutaneous melanoma expresses the pleiotropic factor thrombospondin-1 (TSP-1). Herein, we show that TSP-1 (RNA and protein), undetectable in four cultures of melanocytes and a RGP melanoma, was variously present in 13 cell lines from advanced melanomas or metastases. Moreover, microarray analysis of 55 human lesions showed higher TSP-1 expression in primary melanomas and metastases than in common and dysplastic nevi. In a functional enrichment analysis, the expression of TSP-1 correlated with motility-related genes. Accordingly, TSP-1 production was associated with melanoma cell motility in vitro and lung colonization potential in vivo. VEGF/VEGFR-1 and FGF-2, involved in melanoma progression, regulated TSP-1 production. These factors were coexpressed with TSP-1 and correlated negatively with Slug (SNAI2), a cell migration master gene implicated in melanoma metastasis. We conclude that TSP-1 cooperates with FGF-2 and VEGF/VEGFR-1 in determining melanoma invasion and metastasis, as part of a Slug-independent motility program.
Assuntos
Movimento Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/patologia , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Humanos , Pulmão/patologia , Melanoma/metabolismo , Camundongos , Células NIH 3T3 , Metástase Neoplásica , Fenótipo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição da Família SnailRESUMO
Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. On the basis of its histopathology and molecular-genomic changes, ovarian cancer has been divided into subtypes, each with distinct biology and outcome. The aim of this study was to develop a panel of patient-derived EOC xenografts that recapitulate the molecular and biologic heterogeneity of human ovarian cancer. Thirty-four EOC xenografts were successfully established, either subcutaneously or intraperitoneally, in nude mice. The xenografts were histologically similar to the corresponding patient tumor and comprised all the major ovarian cancer subtypes. After orthotopic transplantation in the bursa of the mouse ovary, they disseminate into the organs of the peritoneal cavity and produce ascites, typical of ovarian cancer. Gene expression analysis and mutation status indicated a high degree of similarity with the original patient and discriminate different subsets of xenografts. They were very responsive, responsive, and resistant to cisplatin, resembling the clinical situation in ovarian cancer. This panel of patient-derived EOC xenografts that recapitulate the recently type I and type II classification serves to study the biology of ovarian cancer, identify tumor-specific molecular markers, and develop novel treatment modalities.
Assuntos
Transplante de Neoplasias/métodos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transplante Heterólogo/métodos , Animais , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário , Cisplatino/farmacologia , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
Taxanes are potent inhibitors of cell motility, a property implicated in their antiangiogenic and antimetastatic activity and unrelated to their antiproliferative effect. The molecular mechanism of this anti-motility activity is poorly understood. In this study, we found that paclitaxel induced tubulin acetylation in endothelial and tumor cells, at concentrations that affected cell motility but not proliferation (10(-8) to 10(-9) M, for 4 hours). Induction of tubulin acetylation correlated with inhibition of motility but not proliferation based on a comparison of highly and poorly cytotoxic taxanes (paclitaxel and IDN5390) and tumor cell lines sensitive and resistant to paclitaxel (1A9 and 1A9 PTX22). Consistent with the hypothesis that tubulin deacetylase activity might affect cell response to the anti-motility activity of taxanes, we found that overexpression of the tubulin deacetylase SIRT2 increased cell motility and reduced cell response to the anti-motility activity of paclitaxel. Conversely, the SIRT2 inhibitor splitomicin reduced cell motility and potentiated the anti-motility activity of paclitaxel. The inhibitory effect was further potentiated by the addition of the HDAC6 inhibitor trichostatin A. Paclitaxel and splitomicin promoted translocation into the nucleus--and hence activation--of FOXO3a, a negative regulator of cell motility. This study indicates a role for SIRT2 in the regulation of cell motility and suggests that therapies combining sirtuin inhibitors and taxanes could be used to treat cell motility-based pathologic processes such as tumor angiogenesis, invasion, and metastasis.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Movimento Celular/efeitos dos fármacos , Sirtuína 2/antagonistas & inibidores , Taxoides/farmacologia , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/genética , Inativação Gênica , Humanos , Microtúbulos/metabolismo , Ligação Proteica , Transporte Proteico , Interferência de RNA , Sirtuína 2/genética , Sirtuína 2/metabolismo , Taxoides/administração & dosagemRESUMO
We previously identified regulator of G-protein signaling 5 (RGS5) among several genes expressed by tumor-derived endothelial cells (EC). In this study, we provide the first in vivo/ex vivo evidence of RGS5 protein in the vasculature of ovarian carcinoma clinical specimens and its absence in human ovaries. Consistent with this, we show higher amounts of Rgs5 transcript in EC isolated from human cancers (as opposed to normal tissues) and demonstrate that expression is sustained by a milieu of factors typical of the proangiogenic tumor environment, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2). Supporting these findings, we show elevated levels of Rgs5 mRNA in the stroma from strongly (as opposed to weakly) angiogenic ovarian carcinoma xenografts and accordingly, we also show more of the protein associated to the abnormal vasculature. RGS5 protein predominantly colocalizes with the endothelium expressing platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) and to a much lesser extent with perivascular/mural cells expressing platelet-derived growth factor receptor-beta (PDGFR-ß) or alpha smooth muscle actin (αSMA). To toughen the relevance of the findings, we demonstrate RGS5 in the blood vessels of other cancer models endowed with a proangiogenic environment, such as human melanoma and renal carcinoma xenografts; to the contrary, it was undetectable in the vasculature of normal mouse tissues. RGS5 expression by the cancer vasculature triggered and retained by the proangiogenic microenvironment supports its exploitation as a novel biomarker and opens the path to explore new possibilities of therapeutic intervention aimed at targeting tumor blood vessels.
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica , Proteínas RGS/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Prognóstico , Proteínas RGS/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We showed previously that GnRH receptors are expressed in melanoma cells; their activation reduces cell growth and metastatic behavior. Here, we investigated whether GnRH agonists might affect the expression of genes involved in melanoma progression. By genome-wide transcriptomic and real-time PCR analysis, we first observed that GnRH agonists decrease the expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) (all isoforms) in BLM melanoma cells. Then, we demonstrated that GnRH agonists specifically decrease the expression of the VEGF165 isoform as well as its secretion from BLM cells. These data suggested that activation of GnRH receptors might reduce the pro-angiogenic behavior of melanoma cells. To verify this hypothesis, we treated BLM cells with a GnRH agonist; the conditioned medium from these cells was tested to assess its capability to stimulate human umbilical vein endothelial cell (HUVEC) motility. The migration of HUVECs towards the conditioned medium of GnRH agonist-treated BLM cells was significantly lower than the migration of HUVECs toward the conditioned medium of untreated cells. Thus, GnRH agonists reduce the pro-angiogenic behavior of melanoma cells through a decreased production of bioactive VEGF. We then found that GnRH receptors are also expressed on HUVECs and that GnRH agonists reduce their ability to proliferate and to form capillary-like tubes when stimulated by VEGF. These findings suggest that GnRH agonists exert an anti-angiogenic activity indirectly by decreasing VEGF secretion from tumor cells and directly by counteracting the pro-angiogenic activity of the growth factor. These data might lead to the development of novel targeted approaches for melanoma.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/agonistas , Melanoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Pamoato de Triptorrelina/análogos & derivados , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/irrigação sanguínea , Melanoma/genética , Melanoma/patologia , Neovascularização Patológica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Pamoato de Triptorrelina/administração & dosagem , Pamoato de Triptorrelina/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Protease-activated receptor-1 (PAR-1) is a unique G-protein-coupled receptor belonging to the protease-activated receptor family. Its activation leads to downstream signaling events that launch a variety of cellular responses related to tumor progression. PAR-1 expression has been associated to a variety of human cancers, and our previous studies reveal a high PAR-1 expression in melanoma specimens as compared to common nevi. In the present study, we investigated the contribution of PAR-1 to the malignant phenotype of human melanoma cell lines obtained from cutaneous primary lesions, capable of different metastatic behaviors in the patients from which they have been derived. We found that melanoma cells isolated from lesions giving rise to metastases in patients (WM115, WM278A, WM1361A, WM983A), had higher PAR-1 mRNA and protein expression, as compared to those obtained from lesions that did not develop metastatic disease (WM793, WM35). The cells isolated from metastatic primary lesions were able to colonize the lungs of immunodeficient SCID mice while those isolated from non-metastatic lesions were not. Additionally, cells expressing elevated PAR-1 had higher migratory and invasive abilities than those holding minimal PAR-1 expression. The migration and invasion capabilities of the melanoma cells expressing high PAR-1 were hampered by genetic and pharmacological interventions. The reduction of PAR-1 expression by siRNA and the inhibition of PAR-1 function by the SCH79797 specific antagonist significantly decreased the melanoma cell motility and invasiveness, down to an extent similar to that of the non-metastatic and low PAR-1 expressing cells. Our results provide strong evidence supporting the implication of PAR-1 in the malignant progression of human melanoma.