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No / The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2- yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 +/- 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

No / Fibroblast growth factor receptors (FGFR) play key roles in proliferation, differentiation, and tumorigenesis. Many urothelial carcinomas contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We examined FGFR expression in telomerase-immortalized normal human urothelial cells, urothelial carcinoma cell lines, and tumor samples and showed that FGFR1 expression is increased in a high proportion of cell lines and tumors independent of stage and grade. To determine the role of FGFR1 in low-stage bladder cancer, we overexpressed FGFR1 in telomerase-immortalized normal human urothelial cells and examined changes in proliferation and cell survival in response to FGF2. FGFR1 stimulation increased proliferation and reduced apoptosis. To elucidate the mechanistic basis for these alterations, we examined the signaling cascades activated by FGFR1. FRS2alpha and PLCgamma were activated in response to FGF2, leading to activation of the mitogen-activated protein kinase pathway. The level of mitogen-activated protein kinase activation correlated with the level of cyclin D1, MCL1, and phospho-BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in urothelial carcinoma cell lines revealed differential FGFR1 dependence. JMSU1 cells were dependent on FGFR1 expression for survival but three other cell lines were not. Two cell lines (JMSU1 and UMUC3) were dependent on FGFR1 for growth in soft agar. Only one of the cell lines tested (UMUC3) was frankly tumorigenic; here, FGFR1 knockdown inhibited tumor growth. Our results indicate that FGFR1 has significant effects on urothelial cell phenotype and may represent a useful therapeutic target in some cases of urothelial carcinoma.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata. / Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata. / Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.