Células epiteliais, endoteliais e fibroblastos podem ser induzidos a apoptose (anoikis) quando se destacam da matriz extracelular. O processo de anoikis tem a função de proteger o organismo da colonização inapropriada destas células. O processo de apoptose pode ser induzido por vários estímulos, dentre eles, o óxido nítrico (NO). O radical livre NO pode ser sintetizado em organismos vivos pela ação das enzimas NO sintases (NOS), como também pode ser liberado por compostos doadores de NO. A geração intracelular ou extracelular de NO pode resultar em diferenciação, proliferação ou morte celular, dependendo da natureza e quantidades de NO gerado no processo, como também do tipo de celular alvo da ação do radical livre. Este trabalho estudou a indução do processo de apoptose iniciada pelo doador de óxido nítrico nitroprussiato de sódio (SNP) em células de linhagem células A431 de carcinoma epidermóide humano. A partir de 6 horas de tratamento com SNP 1mM, as células destacadas já estão positivas para citoqueratina 18 (CK18), anexina-V e apresentam caspases 3 e 8 ativas. Elevado conteúdo de bax e queda de bcl-2, representando uma razão bcl-2/bax compatível com apoptose é alcançada após 24 horas de tratamento com SNP 1mM, bem como ativação de JNK, reação de Tunnel positivo, positividade ao corante Hoescht 33258, atividade insignificante de LD, marcação positiva para anexina-V e negativa para iodeto de propídio (PI). As propriedades antiadesivas do NO foram observadas analisando-se o aumento da perda de aderência e diminuição de viabilidade das células destacadas após tratamento com SNP e positividade para anticorpo anticitoqueratina AE1 e AE2. Células plaqueadas em diferentes superfícies que dificultam o processo de aderência, quando submetidas ao tratamento com SNP rompem mais facilmente os complexos de adesão e se destacam mais rapidamente. Os resultados apresentados sugerem que o NO promove alterações na integridade do citoesqueleto, que induzem as células A431 à apoptose por perda de aderência a matriz extracelular. / Epithelial cells, endotelial cells, and fibroblasts undergo a special kind of apoptosis (anoikis) that occurs when they are displaced from the extracellular matriz. Anoikis is thought to protect tissues from inappropriate colonization. A number of studies described apoptogenic and anti-apoptotic properties for nitric oxide (NO). Our studies focused on the induced apoptosis of the human epithelial carcinoma cell line A431 after exposure to an NO donor, sodium nitroprusside. Initially, the following features were associated with NO-induced A431 cell death: decreased expression of Bcl-2, enhanced expression of Bax, and activation of the stress related MAP kinase, JNK. In addition, NO treated cells were positive for the TUNEL assay, and for Hoescht 33258 staining. Caspase 8 and 3 activities was detected in the system. Continuing our investigation on the mechanisms on the underlying the NO-induced apoptosis, we investigated modifications on the cytoskeleton upon exposure of A431 cells to the NO donor. NO presents anti-adhesive properties, either down regulating the expression of adhesion molecules or disrupting adhesion complexes. Accordingly, we found that A431 cells undergoing NO-induced apoptosis (positive for Anexin V labeling and negative for propidiurn iodide labeling), featured a loss of cytoskeleton integrity, and an insufficient rescue of the supernatant cells. In conclusion, our findings suggest that NO promote the adhesion related apoptosis, anoikis, in A431 cells.

Células epiteliais, endoteliais e fibroblastos podem ser induzidos a apoptose (anoikis) quando se destacam da matriz extracelular. O processo de anoikis tem a função de proteger o organismo da colonização inapropriada destas células. O processo de apoptose pode ser induzido por vários estímulos, dentre eles, o óxido nítrico (NO). O radical livre NO pode ser sintetizado em organismos vivos pela ação das enzimas NO sintases (NOS), como também pode ser liberado por compostos doadores de NO. A geração intracelular ou extracelular de NO pode resultar em diferenciação, proliferação ou morte celular, dependendo da natureza e quantidades de NO gerado no processo, como também do tipo de celular alvo da ação do radical livre. Este trabalho estudou a indução do processo de apoptose iniciada pelo doador de óxido nítrico nitroprussiato de sódio (SNP) em células de linhagem células A431 de carcinoma epidermóide humano. A partir de 6 horas de tratamento com SNP 1mM, as células destacadas já estão positivas para citoqueratina 18 (CK18), anexina-V e apresentam caspases 3 e 8 ativas. Elevado conteúdo de bax e queda de bcl-2, representando uma razão bcl-2/bax compatível com apoptose é alcançada após 24 horas de tratamento com SNP 1mM, bem como ativação de JNK, reação de Tunnel positivo, positividade ao corante Hoescht 33258, atividade insignificante de LD, marcação positiva para anexina-V e negativa para iodeto de propídio (PI). As propriedades antiadesivas do NO foram observadas analisando-se o aumento da perda de aderência e diminuição de viabilidade das células destacadas após tratamento com SNP e positividade para anticorpo anticitoqueratina AE1 e AE2. Células plaqueadas em diferentes superfícies que dificultam o processo de aderência, quando submetidas ao tratamento com SNP rompem mais facilmente os complexos de adesão e se destacam mais rapidamente. Os resultados apresentados sugerem que o NO promove alterações na integridade do citoesqueleto, que induzem as células A431 à apoptose por perda de aderência a matriz extracelular. / Epithelial cells, endotelial cells, and fibroblasts undergo a special kind of apoptosis (anoikis) that occurs when they are displaced from the extracellular matriz. Anoikis is thought to protect tissues from inappropriate colonization. A number of studies described apoptogenic and anti-apoptotic properties for nitric oxide (NO). Our studies focused on the induced apoptosis of the human epithelial carcinoma cell line A431 after exposure to an NO donor, sodium nitroprusside. Initially, the following features were associated with NO-induced A431 cell death: decreased expression of Bcl-2, enhanced expression of Bax, and activation of the stress related MAP kinase, JNK. In addition, NO treated cells were positive for the TUNEL assay, and for Hoescht 33258 staining. Caspase 8 and 3 activities was detected in the system. Continuing our investigation on the mechanisms on the underlying the NO-induced apoptosis, we investigated modifications on the cytoskeleton upon exposure of A431 cells to the NO donor. NO presents anti-adhesive properties, either down regulating the expression of adhesion molecules or disrupting adhesion complexes. Accordingly, we found that A431 cells undergoing NO-induced apoptosis (positive for Anexin V labeling and negative for propidiurn iodide labeling), featured a loss of cytoskeleton integrity, and an insufficient rescue of the supernatant cells. In conclusion, our findings suggest that NO promote the adhesion related apoptosis, anoikis, in A431 cells.

Phosphatidylcholine (PC) is an essential component of biological membranes and is synthesized by the CDP-choline pathway under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCT?). Ras transformed cells have increased lipid synthesis; the aim of this study was to determine if upregulation of CCT? was part of this transformed phenotype. Rat intestinal epithelial cell lines (IEC) and three oncogenic H-ras expressing IEC (IEC-Ras) were used to investigate the role of CCT? and phosphatidylcholine (PC) synthesis in resistance to detachment dependant apoptosis, termed anoikis. IEC-Ras have increased CCT? expression within the nucleus. Reduction of CCT? expression with lentiviral short hairpin RNA sensitized IEC-Ras to anoikis and decreased PC degradation, but did not change PC synthesis. Thus, in addition to CCT? being involved in anoikis-resistance in IEC-Ras these data indicate the possibility that it may also have nuclear-specific functions.

As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains ix, 127 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 108-123).

Head and neck squamous cell carcinoma (HNSCC) develops in mucous membranes that line the mouth, throat and sinuses. It is the sixth most common cancer worldwide with more than 600,000 new cases diagnosed every year. The 5 year survival of HNSCC patients is less than 50% after initial diagnosis, while median survival after relapse or metastasis is less than one year. Unfortunately, the prognosis of HNSCC has not changed in the last two decades, mostly due to the poor understanding of the mechanism of this disease. New therapeutic targets and agents are in urgent need.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Recentemente, nosso grupo demonstrou que a matriz extracelular de astrocitomas promove a seleçãode células endoteliais altamente proliferativas, porém com reduzida capacidade tubulogênica, além de determinar a morte de uma segunda sub-população endotelial, por desaderência ou anoikis. Estratégias de simulação dos teores de tenascina-C (TN-C) e fibronectina (FN) nas matrizes de astrocitomas, realizados com ambas as proteínas purificadas na forma de substratos definidos, sugeriram que o balanço TN-C:FN estava relacionado com os fenótipos endoteliais observados. No entanto, este procedimento não permitia abordar a participação de outros componentes da matriz tumoral nativa neste processo. Com objetivo de estudar a modulação do fenótipo angiogênico das células endoteliais por matrizes de astrocitoma, realizamos o silenciamento da expressão de TN-C na linhagem de astrocitoma U-373 MG. O silenciamento foi confirmado por western blotting, PCR em tempo real e ELISA, que permitiram concluir que, no período pós-transfecção (120h) necessário para se obter matrizes tumorais nativas para ensaios funcionais com células endoteliais, as células U-373 MG mantiveram-se silenciadas em índices superiores a 90%. A diminuição de TN-C nas matrizes tumorais resultou em um pequeno (≅18%, em média), porém significativo aumento na taxa de adesão endotelial. HUVECs incubadas com a matriz secretadas por células silenciadas apresentaram uma redução de ≅35% do número de núcleos picnóticos, quando comparadas a HUVECs incubadas com a matriz de células U-373 MG (selvagens ou transfectadas com siRNA controle). O silenciamento da expressão da TN-C na matriz nas células U-373 MG restaurou ainda o defeito tubulogênico das células endoteliais, que passaram a apresentar formação de tubos comparável à obtida quando HUVECs foram incubadas com sua matriz autóloga, rica em FN. Tais resultados apoiam observações anteriores do grupo, que já sugeriam que a maior proporção de FN na matriz autóloga, comparada a matriz do astrocitoma, seria o fator principal para a seleção dos fenótipos angiogênicos observados, demonstrando mais uma vez a importância do balanço FN:TN-C na regulação de processos angiogênicos. Dados anteriores sugeriam ainda que a sub-população endotelial que morre por anoikisapós contato prolongado (24 horas) com matrizes de astrocitomas corresponde a células que já haviam entrado na fase S do ciclo celular, no início da incubação. A fim de nos aprofundarmos sobre a participação do ciclo celular neste processo, a expressão da proteína p27, um inibidor de quinases dependentes de ciclinas (CKI), também foi analisada. HUVECs incubadas com a matriz de astrocitoma apresentaram um aumento de 2 a 3 vezes na expressão de p27, quando comparada com HUVECs provenientes de sua matriz autóloga. No entanto, células endoteliais incubadas com matriz secretada por células U-373 MG silenciadas apresentaram um nível de expressão de p27 comparável ao das HUVECs incubadas com matriz secretada por células selvagens, indicando que a expressão de TN-C não modula, ou não está diretamente correlacionada à expressão da proteína p27. Este resultado sugere que outros componentes da matriz tumoral devam estar envolvidos na modulação do ciclo celular endotelial. / We have previously shown that extracellular matrices secreted by high-grade astrocitoma promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells, while also leading to the death, by anoikis, of another endothelial subpopulation. The use of defined adhesion substrata containing various levels of tenascin-C (TN-C) and fibronectin (FN) allowed us to confirm that the balance between TN-C and FN was a major cause of the selection of both endothelial phenotypes. However, this strategy did not allow us to address the potential role of other molecular components present in tumor matrices. In the present work, we studied the effect of a matrix produced by U-373 MG cells previously silenced for TN-C expression in endothelial cell adhesion, survival and tubulogenic differentiation, as compared to the matrix secreted by wild-type astrocitoma cells. U-373 MG cells silencing was confirmed by Western blotting, real time RT-PCR and ELISA, and cells remained silenced (≥ 90 %) throughout the 120 hours period necessary for generating immobilized native matrices required for endothelial cell function assays. Human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix secreted by astrocitoma cells silenced for TN-C expression was significantly increased by ≅18 %, as compared to wild-type tumor matrix. The number of HUVECs exhibiting picnotic nuclei a hallmark of advanced apoptosis has also been significantly decreased by ≅35 %, when endothelial cells were allowed to incubate with TN-C-depleted tumor matrix, as compared to the wild-type tumor matrix. Concerning angiogenic differentiation, endothelial cells incubated with the matrix produced by silenced U-373 cells were strongly attenuated for their tubulogenesis defect, as compared to HUVECs incubated with the TN-C-rich wild-type matrix. Thus, these data corroborated our previous observations that TN-C in astrocitoma matrices crucially interferes with endothelial cell differentiation. Besides adhesion, survival and tubulogenic differentiation, the responses of endothelial cells to astrocitomas matrices are also affected by cell cycle transitions. We have previously shown that endothelial cells undergoing anoikis had already transitioned through the S-phase of cell cycle at the moment of seeding. Thus, we decided to investigate the expression of p27 protein, an inhibitor of ciclin-dependent kinases (CKI) that has been already implicated in glioma angiogenesis. We found that HUVECs incubated with the matrix secreted by U-373 MG wild-type cells for 24 hours exbibited a 2 to 3-fold increase in p27 expression. In contrast to the other results discussed herein, these differences were not correlated with the expression of TN-C by U-373 MG cells, since the matrix produced by tumor cells silenced for TN-C did not alter the expression of p27 in endothelial cells. Overall, the present data suggest that, although TN-C in native tumor matrix does play a major role in endothelial cell angiogenic differentiation, other matrix components may act in concert with TN-C to modulate endothelial cell proliferation in tumor contexts.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Recentemente, nosso grupo demonstrou que a matriz extracelular de astrocitomas promove a seleçãode células endoteliais altamente proliferativas, porém com reduzida capacidade tubulogênica, além de determinar a morte de uma segunda sub-população endotelial, por desaderência ou anoikis. Estratégias de simulação dos teores de tenascina-C (TN-C) e fibronectina (FN) nas matrizes de astrocitomas, realizados com ambas as proteínas purificadas na forma de substratos definidos, sugeriram que o balanço TN-C:FN estava relacionado com os fenótipos endoteliais observados. No entanto, este procedimento não permitia abordar a participação de outros componentes da matriz tumoral nativa neste processo. Com objetivo de estudar a modulação do fenótipo angiogênico das células endoteliais por matrizes de astrocitoma, realizamos o silenciamento da expressão de TN-C na linhagem de astrocitoma U-373 MG. O silenciamento foi confirmado por western blotting, PCR em tempo real e ELISA, que permitiram concluir que, no período pós-transfecção (120h) necessário para se obter matrizes tumorais nativas para ensaios funcionais com células endoteliais, as células U-373 MG mantiveram-se silenciadas em índices superiores a 90%. A diminuição de TN-C nas matrizes tumorais resultou em um pequeno (≅18%, em média), porém significativo aumento na taxa de adesão endotelial. HUVECs incubadas com a matriz secretadas por células silenciadas apresentaram uma redução de ≅35% do número de núcleos picnóticos, quando comparadas a HUVECs incubadas com a matriz de células U-373 MG (selvagens ou transfectadas com siRNA controle). O silenciamento da expressão da TN-C na matriz nas células U-373 MG restaurou ainda o defeito tubulogênico das células endoteliais, que passaram a apresentar formação de tubos comparável à obtida quando HUVECs foram incubadas com sua matriz autóloga, rica em FN. Tais resultados apoiam observações anteriores do grupo, que já sugeriam que a maior proporção de FN na matriz autóloga, comparada a matriz do astrocitoma, seria o fator principal para a seleção dos fenótipos angiogênicos observados, demonstrando mais uma vez a importância do balanço FN:TN-C na regulação de processos angiogênicos. Dados anteriores sugeriam ainda que a sub-população endotelial que morre por anoikisapós contato prolongado (24 horas) com matrizes de astrocitomas corresponde a células que já haviam entrado na fase S do ciclo celular, no início da incubação. A fim de nos aprofundarmos sobre a participação do ciclo celular neste processo, a expressão da proteína p27, um inibidor de quinases dependentes de ciclinas (CKI), também foi analisada. HUVECs incubadas com a matriz de astrocitoma apresentaram um aumento de 2 a 3 vezes na expressão de p27, quando comparada com HUVECs provenientes de sua matriz autóloga. No entanto, células endoteliais incubadas com matriz secretada por células U-373 MG silenciadas apresentaram um nível de expressão de p27 comparável ao das HUVECs incubadas com matriz secretada por células selvagens, indicando que a expressão de TN-C não modula, ou não está diretamente correlacionada à expressão da proteína p27. Este resultado sugere que outros componentes da matriz tumoral devam estar envolvidos na modulação do ciclo celular endotelial. / We have previously shown that extracellular matrices secreted by high-grade astrocitoma promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells, while also leading to the death, by anoikis, of another endothelial subpopulation. The use of defined adhesion substrata containing various levels of tenascin-C (TN-C) and fibronectin (FN) allowed us to confirm that the balance between TN-C and FN was a major cause of the selection of both endothelial phenotypes. However, this strategy did not allow us to address the potential role of other molecular components present in tumor matrices. In the present work, we studied the effect of a matrix produced by U-373 MG cells previously silenced for TN-C expression in endothelial cell adhesion, survival and tubulogenic differentiation, as compared to the matrix secreted by wild-type astrocitoma cells. U-373 MG cells silencing was confirmed by Western blotting, real time RT-PCR and ELISA, and cells remained silenced (≥ 90 %) throughout the 120 hours period necessary for generating immobilized native matrices required for endothelial cell function assays. Human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix secreted by astrocitoma cells silenced for TN-C expression was significantly increased by ≅18 %, as compared to wild-type tumor matrix. The number of HUVECs exhibiting picnotic nuclei a hallmark of advanced apoptosis has also been significantly decreased by ≅35 %, when endothelial cells were allowed to incubate with TN-C-depleted tumor matrix, as compared to the wild-type tumor matrix. Concerning angiogenic differentiation, endothelial cells incubated with the matrix produced by silenced U-373 cells were strongly attenuated for their tubulogenesis defect, as compared to HUVECs incubated with the TN-C-rich wild-type matrix. Thus, these data corroborated our previous observations that TN-C in astrocitoma matrices crucially interferes with endothelial cell differentiation. Besides adhesion, survival and tubulogenic differentiation, the responses of endothelial cells to astrocitomas matrices are also affected by cell cycle transitions. We have previously shown that endothelial cells undergoing anoikis had already transitioned through the S-phase of cell cycle at the moment of seeding. Thus, we decided to investigate the expression of p27 protein, an inhibitor of ciclin-dependent kinases (CKI) that has been already implicated in glioma angiogenesis. We found that HUVECs incubated with the matrix secreted by U-373 MG wild-type cells for 24 hours exbibited a 2 to 3-fold increase in p27 expression. In contrast to the other results discussed herein, these differences were not correlated with the expression of TN-C by U-373 MG cells, since the matrix produced by tumor cells silenced for TN-C did not alter the expression of p27 in endothelial cells. Overall, the present data suggest that, although TN-C in native tumor matrix does play a major role in endothelial cell angiogenic differentiation, other matrix components may act in concert with TN-C to modulate endothelial cell proliferation in tumor contexts.

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 encourages the production of the pro-metastatic isoform of CPEB2, which contributes the aggressive phenotype of the tumor. We utilized numerous methods to measure the metastatic function of cultured TNBC cells to determine if SRSF3 strongly promoted the metastatic function. These data showed that siRNA reduction of SRSF3 was able to reduce the metastatic potential of cancer cells. These findings suggest that SRSF3 has great potential as a therapeutic measure to reduce and minimize the aggressiveness of TNBC tumors.