Vulnerabilidades específicas de células malignas humanas dependentes de Ras oncogênico: FGF2 e PMA como supressores de tumor / Specific vulnerabilities of human malignant cells dependent on oncogenic Ras: FGF2 and PMA as tumor suppressors

Um passo limitante no desenvolvimento de fármacos para terapias do câncer está na descoberta de vulnerabilidades específicas de células tumorais que sirvam à identificação de alvos moleculares apropriados à intervenção farmacológica. Esta é a motivação central desta tese, cuja abordagem experimental focaliza a ação oncogênica das proteínas Ras. Amplificação ou mutação ativadora nos proto-oncogenes ras estão entre as alterações genéticas mais frequentes em cânceres. Essas lesões genéticas aparecem na origem etiológica de múltiplas formas de fenótipos malignos. Mas, essas lesões oncogênicas também conferem susceptibilidades letais às células malignamente transformadas frente a determinados agentes que não interferem significativamente nas funções vitais de células normais. Nos últimos anos nosso laboratório vem estudando os mecanismos moleculares da ação antiproliferativa do fator de crescimento FGF2 (Fibroblast Growth Factor2) e do éster de forbol PMA (Phorbol-12-Myristate-13-Acetate) em linhagens de células murinas malignas dependentes de ras oncogênico. Nesta tese investigamos quanto de nossas observações anteriores com células murinas são aplicáveis a células humanas. Nesse sentido focalizamos a linhagem HaCaT de queratinócitos humanos imortalizados e seus subclones malignizados por expressão ectópica de H-RasV12; além disso, numa triagem inicial também examinamos treze linhagens celulares humanas derivadas de tumores naturais portadores de mutação ativadora em H-Ras, N-Ras ou K-Ras. Nossos resultados mostram que os queratinócitos da linhagem parental HaCaT expressam receptores de FGFs e respondem mitogenicamente tanto a FGF2 como a PMA; portanto, ambos FGF2 e PMA são benéficos aos queratinócitos HaCaT. Por outro lado, o FGF2 mostrou-se citotóxico para subclones HaCaT que expressam H-RasV12 induzível, mas sublinhagens HaCaT com expressão constitutiva de H-RasV12 mostraram-se resistentes à ação citotóxica de FGF2. Diferentemente de FGF2, PMA bloqueou a proliferação de sublinhagens clonais HaCaT-H-RasV12 em ambos substrato sólido e suspensão de agarose e, também, reduziu a estratificação dos queratinócitos HaCaT-H-RasV12 em culturas organotípicas. PMA foi citotóxico e não citostático, pois induziu morte apoptótica sem causar arresto em nenhuma fase específica do ciclo celular. Em HaCaT parental, PMA induziu aumento transitório dos níveis intracelulares de espécies reativas de oxigênio (ROS), mas nos queratinócitos HaCaT-H-RasV12, PMA causou aumentos mais altos e persistentes de ROS, o que promove forte estresse oxidativo, provavelmente responsável pela toxidez deste ester de forbol. Entre as treze linhagens celulares humanas malignas com H-Ras, N-Ras ou K-Ras mutados, onze foram vulneráveis à ação citotóxica de PMA; mas apenas uma delas, a linhagem de tumor urotelial UM-UC-3, foi sensível ao efeito anti-proliferativo de FGF2. Em conclusão, células malignas humanas com Ras mutado parecem superar rapidamente uma possível toxidez de FGF2, mas não ultrapassam a toxidez causada por PMA

A challenge in drug development for cancer therapy is the discovering of molecular targets suitable for pharmacological interference. This challenge was the main motivation of the present thesis. Amplification or activating mutation in ras proto-oncogenes are among the most frequent genetic lesions in human cancer. Actually, mutated Ras onco-proteins are in the etiological roots of multiple malignant phenotypes; however these onco-proteins also cause specific lethal vulnerabilities even in robust malignant cells. Recently, our laboratory reported that malignant murine cell lines dependent on oncogenic Ras are prone to toxicity initiated by FGF2 (Fibroblast Growth Factor 2) and PMA (Phorbol-12-Myristate-13-Acetate), which are not harmful to normal cells. This cytotoxicity of FGF2 and PMA very likely follows different molecular mechanisms, which, however, are not yet completely understood. The aim of this thesis was to investigate whether these vulnerabilities found in murine malignant cells were also valid for human malignant cell lines dependent on oncogenic Ras. To this end the experimental approach was focused on the HaCaT cell line of immortalized human keratinocytes and its sublines transformed by H-RasV12 ectopic expression. In addition thirteen human cell lines derived from natural tumor carrying mutated H-Ras, N-Ras or K-Ras oncogenes were also screened. The results showed that HaCaT keratinocytes express FGF receptors and respond mitogenically to both FGF2 and PMA. On the other hand, FGF2 was cytotoxic to HaCaT subclones expressing inducible H-RasV12. But, HaCaT sublines constitutively expressing H-RasV12 were resistant to FGF2 toxicity. However, PMA was toxic to all HaCaT-H-RasV12 sublines, inhibiting proliferation in both solid substrate and agarose suspension cultures and, also reducing stratification in organotypic cultures. Furthermore, in HaCaT-H-RasV12 sublines, but not in the parental HaCaT line, PMA caused a persistently high increase in intracellular levels of reactive oxygen species (ROS) and concomitantly induced apoptosis. Moreover, eleven of the thirteen human tumor cell lines with mutated H-Ras, N-Ras or K-Ras, were growth inhibited by PMA, whereas only one of them was inhibited by FGF2, the urothelial tumor cell line UM-UC-3. In conclusion, human malignant cells driven by Ras oncogenes very likely rapidly overcome FGF2 toxicity, whereas they remain stably vulnerable to PMA cytotoxicity

Acinar Cell Carcinoma of the Pancreas: A Report of Two Cases with Long-term Follow-up and a Review of the Literature

Acinar cell carcinoma (ACC) of the pancreas is a rare malignancy making up approximately 1% of pancreatic non-endocrine malignant tumors. The common finding on computed tomography is a solitary, well-defined, heterogenous hypodense mass with enhancing capsule. ACC is a highly cellular tumor with minimal stroma and a lack of stromal desmoplasia. The accurate diagnosis of ACC cannot typically be done by histology alone but rather requires immunohistochemical staining or electron microscopy for the identification of pancreatic enzymes and zymogen granules. ACC has been considered a cancer with a poor prognosis due to frequent metastasis, a high recurrence rate, and low respectability. Surgical resection is the treatment of choice that can lead to long-term survival. ACC has a better prognosis than ductal carcinoma of the pancreas, but a worse prognosis compared to islet cell carcinoma. We report two cases of ACC with 5-year survival after surgical resection.

Management of nonfunctioning islet cell tumors of the pancreas / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To analyze the clinical and pathological features in order to investigate appropriate way of diagnosis and treatment for non-functional islet cell tumors of the pancreas (NFICT).</p><p><b>METHODS</b>The data and experience of surgically treated 43 patients with pathologically confirmed NFICT over the last 30 years were retrospectively reviewed. The survival rate was estimated using Kaplan-Meier method and the potential risk factors affecting survival were compared with Log rank test.</p><p><b>RESULTS</b>There were 7 males and 36 females in this series with a mean age of 31.6 years ranged from 8 to 67 years. Twenty-eight patients were diagnosed as having non-functional islet cell carcinomas of the pancreas (NFICC) and 15 patients benign islet cell tumors. The most common symptoms in NFICT were abdominal pain 55.8%, nausea and/or vomiting (32.6%), fatigue (25.6%) and abdominal mass (23.3%). Preoperatively, all of those were found to have a mass in their pancrease by ultrasonic and computed tomography examination, with 21 in the head, 10 in the body and 6 in the tail of the pancreas. Multicemtric tumor were found in one patient. Thirty-nine of these 43 patients (90.7%) underwent surgical resection, with a curative resection in 30 (69.8%) and palliative in 9 (20.9%). The resectability and curative resection rate in 28 patients with nonfunctioning islet cell carcinomas of the pancreas was 78.6% and 60.7%, respectively. None of the 15 patients with benign nonfunctioning islet cell tumor of the pancreas died of this disease. While the overall cumulative 5- and 10-year survival rate in 28 patients with non-functional islet cell carcinomas of the pancreas was only 58.1% and 29.0%, respectively. Curative resection, female, younger than 30 years old and mass diameter < 10 cm were found to be positive prognostic factors. But multivariate Cox regression analysis indicated that radical resection was the only independent prognostic factor (P = 0.007).</p><p><b>CONCLUSION</b>Nonfunctioning islet cell tumor of the pancreas is frequently found in young female. Surgical resection, especially curative resection can achieve satisfactory long-term survival.</p>

Neuroendocrine tumors of the pancreas.

Pancreatic Neuroendocrine tumors (PNET) are rare tumors that require a high degree of suspicion for timely diagnosis. They are best divided into functional and nonfunctional varieties. Functional tumors often are symptom specific and are diagnosed at an earlier stage than nonfunctional tumors. The severity of symptoms and pace of disease should dictate therapy. Surgical extirpation remains the only curative modality for localized disease, but palliation of hormone-related symptoms can be achieved with different modalities (Management of hormonal Excess, biologic therapy, chemotherapy and biochemotherapy, local-regional therapy with hepatic arterial embolization).

A Case of Nonfunctioning Pancreatic Islet Cell Carcinomas in Adolescence

Neuroendocrine pancreatic tumors (NPTs) arise from the pancreatic islet cells and belong to the amine and precursor uptake and decarboxylation (APUD) system. These tumors are rare and account for only 1% to 5% of pancreatic tumor. The pancreas is an extremely uncommon site of neoplasia in children and adolescents. For this reason, our understanding of these tumors is still quite limited. Although the complete surgical resection is the key to successful management of all malignant adolescence pancreatic tumors, the information on the possible role of chemotherapy and radiation in recurrent, unresectable, or metastatic cases is purely anecdotal. The 17-year-old man transferred to our hospital with abnormal ultrasonographic findings. Result of abdominal ultrasonographic examination showed a mass in the upper abdomen. He presented with 6months history of intermittent abdominal pain and vomiting and diarrhea. A computed tomography (CT) scan and magnetic resonance showed a 4.5 x 6 cm mass in the head of the pancreas. An ultrasound-guided core biopsy confirmed an pancreatoblastoma or pancreas islet cell tumor. On operative findings, there was locally advanced, unresectable tumor within the pancreatic head. We report a 17-years-old man patient with non-functional panceratic islet cell carcinoma.

Diagnosis and treatment of malignant pancreatic endocrine tumour / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To summarize our experience in the diagnosis and treatment of malignant pancreatic endocrine tumour.</p><p><b>METHODS</b>We retrospectively reviewed 36 cases of malignant pancreatic endocrine tumours in our hospital from July 1987 to April 2002, and summarized its clinical features.</p><p><b>RESULTS</b>Liver metastasis was the main malignant manifestation of malignant pancreatic endocrine tumours (incidence rate 72.2%). Removals of primary lesion and isolated hepatic metastatic lesion were means of curative therapy. Interventional chemotherapy was an important adjuvant treatment.</p><p><b>CONCLUSION</b>Comprehensive therapy plays an important role in improving the prognosis of malignant pancreatic endocrine tumour.</p>

Nonfunctioning islet cell tumor presenting with ascites and portal hypertension.

Nonfunctioning islet cell tumors commonly cause no symptoms. A 22-year-old woman presented with lump in the left hypochondrium, refractory high-protein ascites and evidence of left-sided portal hypertension. At exploratory laparotomy, a 30 cm x 15 cm mass was seen at the splenic hilum, with large collateral vessels around. Distal pancreatectomy with splenectomy was done. Histology of the mass showed malignant islet cell tumor infiltrating the spleen. The patient died in the postoperative period.

Mixed acinar-endocrine carcinoma of the pancreas: a case report

A case of pancreatic carcinoma with both acinar and endocrine features is presented. The patient was a 52-year-old female presenting with jaundice of 3 weeks' duration. The tumor was a 6 x 6 cm-sized round solid mass in the head of pancreas, invading the superior mesenteric vein. Histologically, it was composed of monotonous ovoid cells with eosinophilic granular cytoplasm in solid nests and sheets with occasional acinar and glandular differentiation. Immunohistochemical study revealed coexpression of acinar and endocrine markers; amylase, chromogranin, neuron-specific enolase, glucagon, somatostatin, and gastrin in tumor cells. This is the first documented case of mixed acinar-endocrine carcinoma of the pancreas in Korea, and its amphicrine nature reflects a close histogenetic relationship between pancreatic exocrine and endocrine cells.