RESUMEN
Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3ß protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Meduloblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Caenorhabditis elegans , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Cisplatino/farmacología , Cisplatino/uso terapéutico , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiofenos/farmacología , Tiofenos/uso terapéutico , Pruebas de Toxicidad , Urea/análogos & derivados , Urea/farmacología , Urea/uso terapéuticoRESUMEN
Cisplatin (CisPt) is frequently used in the therapy of urothelial carcinoma (UC). Its therapeutic efficacy is limited by inherent or acquired drug resistance. Here, we comparatively investigated the CisPt-induced response of two different parental urothelial carcinoma cell lines (RT-112, J-82) with that of respective drug resistant variants (RT-112R, J-82R) obtained upon month-long CisPt selection. Parental RT-112 cells were ~2.5 fold more resistant to CisPt than J-82 cells and showed a different expression pattern of CisPt-related resistance factors. CisPt resistant RT-112R and J-82R variants revealed a 2-3-fold increased CisPt resistance as compared to their corresponding parental counterparts. Acquired CisPt resistance was accompanied by morphological alterations resembling epithelial mesenchymal transition (EMT). RT-112R cells revealed lower apoptotic frequency and more pronounced G2/M arrest following CisPt exposure than RT-112 cells, whereas no differences in death induction were observed between J-82 and J-82R cells. CisPt resistant J-82R cells however were characterized by a reduced formation of CisPt-induced DNA damage and related DNA damage response (DDR) as compared to J-82 cells. Such difference was not observed between RT-112R and RT-112 cells. J-82R cells showed an enhanced sensitivity to pharmacological inhibition of checkpoint kinase 1 (Chk1) and, moreover, could be re-sensitized to CisPt upon Chk1 inhibition. Based on the data we suggest that mechanisms of acquired CisPt resistance of individual UC cells are substantially different, with apoptosis- and DDR-related mechanisms being of particular relevance. Moreover, the findings indicate that targeting of Chk1 might be useful to overcome acquired CisPt resistance of certain subtypes of UC.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/genética , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma de Células Transicionales/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Humanos , Transducción de Señal/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/efectos de los fármacos , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
BACKGROUND: Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. METHODS: We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. RESULTS: CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. CONCLUSIONS: There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.
Asunto(s)
Antineoplásicos/toxicidad , Cisplatino/toxicidad , Daño del ADN , Glomérulos Renales/efectos de los fármacos , Túbulos Renales/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Células Cultivadas , Aductos de ADN/efectos de los fármacos , Aductos de ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Túbulos Renales/citología , Túbulos Renales/metabolismo , Compuestos Organoplatinos/toxicidad , Oxaliplatino , RatasRESUMEN
Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes.
Asunto(s)
Empalme Alternativo , Biomarcadores de Tumor/genética , Neoplasias de la Próstata/genética , Proteínas/genética , Anciano , Área Bajo la Curva , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/orina , Biopsia , Línea Celular Tumoral , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Calicreínas/sangre , Masculino , Persona de Mediana Edad , Mucoproteínas , Proteínas Oncogénicas , Proyectos Piloto , Valor Predictivo de las Pruebas , Pronóstico , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/orina , ARN Mensajero/orina , Curva ROCRESUMEN
Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.
Asunto(s)
Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/patología , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: AGR2 is a member of the endoplasmatic reticulum protein disulphide isomerase gene family implicated in tumor metastasis. Its expression pattern, function, and utility as a marker remains to be further investigated. METHODS: Using real-time RT-PCR and immunohistochemistry, changes of expression in different tumor stages were explored in microdissected tumor samples. AGR2 transcript level in urine sediments was scrutinized for suitability as a tumor marker. AGR2 androgen regulation and function were analyzed in cellular prostate cancer models. RESULTS: AGR2 is highly expressed in prostate cancer compared to benign tissue in particular also in low-grade tumors and PIN lesions. AGR2 transcripts were detected in urine sediments of patients undergoing prostate biopsy with significantly higher levels in tumor patients. The urine AGR2/PSA transcript ratio allowed much better discrimination between cancer and benign patients than serum total PSA or %freePSA. Prostate tumor cells express and secrete variable amounts of AGR2 protein, the highest level was found in PC3 cells. In androgen receptor-positive cell lines AGR2 is upregulated by androgens. Increased expression enhanced the migratory and invasive potential but decreased growth and proliferation in vitro and in vivo. CONCLUSION: AGR2 enhances the invasion phenotype of prostate cancer cells while at the same time attenuating cell-cycle progression. This function, its expression pattern and the increased level of AGR transcripts in urine sediments of prostate cancer patients call for further exploration as a prostate cancer marker and a modulator of tumor growth and invasion.