TREAT LIVER DISEASES BY TARGETING HEPATOCYTE NECROPTOSIS

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miRNA-143 and miRNA-145 modulation in colon cancer : from anticancer activity to drug sensitization

Publication . Gomes, Sofia Estevão; Rodrigues, Cecília M. P., 1968-; Borralho, Pedro Miguel Martinho, 1979-

Overall survival of patients with colon cancer has slightly increased over the past 15 years due to the introduction of novel therapies and screening tests in clinical practice. However, mortality from colon cancer remains high, being the fourth most common cause of cancer-related death worldwide. Although most patients are initially responsive to several chemotherapy regimens, the effectiveness of the current systemic therapies in patients with advanced colon cancer remains limited by drug resistance. Therefore, increasing drug sensitivity is a key step towards more effective therapeutic strategies to treat cancer patients. Recent discoveries have revealed that microRNAs (miRNAs/miRs) hold great promise to render tumours responsive to chemotherapy and targeted therapy. In fact, miRNA expression profiles in tumour cells have been shown to promote anti-cancer therapy resistance, by abnormally regulating the expression of genes involved in cellular processes, such as apoptotic responses and redox-mediated mechanisms. Therefore, the studies included in the present thesis were conducted with the purpose of further characterizing the molecular mechanisms by which the tumour suppressors miR-143 and miR-145 regulate anticancer activity and drug sensitization of colon cancer cells. In initial studies, we evaluated the signalling pathways and players modulated by miR-143 and miR-145 expression in colon cancer cells. Functional proteomic analysis revealed that miR-143 and miR-145 modulate several proteins associated with regulation of oxidative stress, cell death and protein folding. In particular, the antioxidant enzyme SOD1 was among the identified proteins whose steady state levels were downregulated by either miR-143 or miR-145. In addition, forced miR-143 expression strongly increased endogenous production of reactive oxygen species (ROS) production, which was completely abrogated by re-introduction of SOD1. Importantly, miR-143 overexpression increased oxaliplatin-induced apoptosis associated with ROS generation, which was abrogated by genetic and pharmacological inhibition of oxidative stress. Therefore, our data provide evidence to support a role for miR-143-induced ROS in mediating cell sensitivity to oxaliplatin. Next, considering the involvement of miR-143 and miR-145 in the EGFR/RAS/MAPK signalling network, we hypothesized that miR-143 and miR-145 play a role in the regulation of colon cancer cellular sensitivity to the anti-EGFR antibody, cetuximab. Our data show that overexpression of miR-143 and miR-145 reduced cell proliferation and migration, sensitized both mutant and wild-type KRAS cells to cetuximab, and strongly potentiated the antitumour effects of cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC). These changes were associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression. Interestingly, we observed that the mechanism underlying cetuximab-mediated ADCC proceeds through a granzyme Bdependent pathway, as granzyme B inhibition abrogated cell death induced by cetuximabmediated ADCC, as well as caspase-3/7 activity. These results further suggest that miR-143 and miR-145 re-sensitize colon cancer cells to cetuximab by stimulating cetuximabdependent ADCC to induce cell death. Finally, we suggest that miR-145 inhibits cancer stem-like cell characteristics, including colon sphere formation and propagation, as well as the expression of specific stem cell markers. Notably, augmented levels of miR-145 in colon cancer stem-like cells increased a panel of well-established differentiation markers, suggesting that miR-145 may contribute to the induction of colon cancer stem-like cell differentiation. In addition, miR-145 reversed chemoresistance by reducing cell viability of colon spheres exposed to 5- fluorouracil-based chemotherapeutic regimens, and undergoing efficient apoptosis elicited by chemotherapy. Overall, our findings underscore the importance of miR-143 and miR-145 in colon cancer, expanding their roles as pro-apoptotic and chemosensitizer components. miR-145 additionally regulates colon cancer stem cells. Importantly, replenishing such tumour suppressor miRNAs in combination with anti-cancer therapy should be regarded and further studied as potential therapeutic options for colon cancer.

2017Doctoral thesis

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Activation of necroptosis in human and experimental cholestasis

Publication . Afonso, Marta; Rodrigues, Pedro; Simão, André; Ofengeim, Dimitry; Carvalho, Tânia; Amaral, Joana D.; Gaspar, Maria Manuela; Cortez-Pinto, Helena; Castro, Rui E.; Yuan, Junying; Rodrigues, Cecília M. P.

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3-/-) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3-/- mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

2016Journal article

Open access

Exploring mek5-erk5 signaling in colorectal cancer : A therapeutic target for chemosensitization

Publication . Pereira, Diane M.; Rodrigues, Cecília M. P., 1968-; Borralho, Pedro Miguel Martinho, 1979-

Colorectal cancer remains one of the leading causes of cancer morbidity and mortality in the world. Despite significant advances in early detection and multimodal care, drug resistance, recurrence, and metastasis persist as major challenges in the management of the disease, calling for the identification of alternative targets for therapeutic intervention. Interestingly, in human colorectal adenomas and adenocarcinomas, there is ample evidence that aberrant MEK5-ERK5 expression and/or activation correlates with tumor-node-metastasis stage. As such, the studies presented in this thesis were conducted with the purpose of defining the contribution of ERK5-mediated signaling to the regulation of hallmark traits associated with disease progression, namely tumor cell resistance to 5-fluorouracil (5-FU), the cornerstone of systemic colorectal cancer treatment; and malignant stem cell-like pools, major players in therapy-refractory disease. Using colorectal cancer cell lines as model, we showed that exposure to 5-FU impairs endogenous MEK5-ERK5 signaling, whereas ectopic expression of a constitutively active MEK5 increases resistance to this drug. Conversely, blocking ERK5 in combination with 5-FU results in impaired tumor cell survival and growth in vitro and in subcutaneous xenografts. Further studies established that ERK5 inhibition promotes 5-FU–induced apoptosis in a TP53-wild-type but not a TP53-null background, implying a p53-dependent axis mediating 5-FU sensitization. In parallel, we demonstrated that MEK5-ERK5 phosphorylation levels are increased in three-dimensional sphere cultures enriched for neoplastic stem-like cells. Further, targeting ERK5 suppresses the rates of tumorsphere formation and the expression/activity of representative markers of immature cancer cell fractions, while sensitizing to 5-FU–based chemotherapy. Moreover, downregulation of NF-κB–mediated IL-8 expression might be a crucial event for the impact of ERK5 inhibition on malignant stem-like phenotypes. Finally, analysis of publicly available databases revealed that increased ERK5 expression correlates with shorter overall survival in colorectal cancer patients, reinforcing the clinical relevance of the MEK5-ERK5 axis. Overall, our findings indicate that upregulated MEK5-ERK5 signaling in colorectal carcinoma cells contributes to a shift to an undifferentiated state, whilst providing a route for cancer (stem) cells to escape cytotoxic insults inflicted by classical chemotherapy, therefore encouraging future investigations on the translational potential of ERK5-targeted agents for antineoplastic treatment and chemosensitization.

2019Doctoral thesis

Restricted access

Targeting hepatocyte necroptosis to treat liver diseases

Publication . Afonso, Marta; Rodrigues, Cecília M. P., 1968-; Castro, Rui Eduardo Mota, 1978-

Chronic liver diseases are considered a major public health concern, and their prevalence and incidence are increasing with serious long-term implications, such as cirrhosis, hepatocellular carcinoma (HCC) and, ultimately, premature death. In particular, non-alcoholic fatty liver disease (NAFLD) and chronic cholestatic liver disease are two distinct pathologic conditions sharing a progressive nature and lacking effective medical therapies. Persistent cell death represents a dominant trigger for chronic inflammation, fibrosis and compensatory cell proliferation, increasing the risk of cancer. Of note, necroptosis or regulated necrosis, which depends on kinase activity of receptor-interacting protein 3 (RIP3), was recently described as novel immunogenic type of cell death that may pathologically impinge on inflammation-driven liver diseases. The main goal of the work presented in this thesis was to evaluate the role of necroptosis in human and experimental models of NAFLD and cholestatic liver disease, and to test whether its inhibition could provide potential therapeutic benefits. First, the role of necroptosis in human and experimental NAFLD was evaluated. We showed that hepatic necroptosis is triggered in NAFLD patients and in two dietary models of non-alcoholic steatohepatitis (NASH), playing a role in disease progression. Indeed, inhibition of necroptosis through genetic ablation of Rip3 attenuated liver injury, steatosis, inflammation, fibrosis and oxidative stress in mice fed a methionine- and choline-deficient NASH-inducing diet. In turn, tumor necrosis factor-α (TNF-α) was shown to trigger RIP3-dependent oxidative stress during hepatocyte necroptosis. We next explored the role of RIP3-dependent signaling in NAFLD-associated HCC using the choline-deficient L amino acid-defined (CDAA) diet murine model. Remarkably, despite associating with increased insulin resistance and hepatic steatosis, absence of RIP3 decreased long-term inflammation and fibrosis, compensatory proliferation of hepatocytes, oxidative stress, genetic cell death resistance in dysplastic hepatocytes, and tissue microenvironment alterations closely associated with NAFLD-driven hepatocarcinogenesis. In parallel, we evaluated the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Our results showed that necroptosis is triggered in the liver of primary biliary cholangitis patients (PBC), mediating hepatic necro-inflammation induced by common bile duct ligation (BDL) in mice. However, deletion of Rip3 failed to prevent BDL-induced secondary biliary fibrosis and was associated with enhanced chronic cholestasis and hepatic iron accumulation, through up-regulation of heme oxygenase-1. As an alternative putative therapeutic approach, we next evaluated whether ablation of microRNA-21 (miR-21) could simultaneously prevent necroptosis and fibrosis in BDL mice. Functional studies established the association between miR-21, its target cyclin dependent kinase 2 associated protein 1 (CDK2AP1) and necroptosis. Further, miR-21 was found increased in the liver of PBC patients and BDL mice, whereas deletion of miR-21 attenuated necroptosis, liver damage, oxidative stress and expression of pro-fibrogenic genes in BDL mice. miR-21 ablation further improved BDL-induced adaptive responses in bile acid homeostasis. Taken together, our studies highlight that inhibition of necroptosis may, at least partially, inhibit NAFLD and NAFLD-associated HCC, as well as cholestatic liver disease pathogenesis. miR-21 inhibition reduces necroptosis and may also afford complementary protective effects. A broader understanding of necroptosis and its intricate role in liver pathophysiology is likely to provide a rationale for the development of therapeutic strategies for NAFLD and cholestatic liver disease based on the targeting of necroptosis-associated signaling pathways.

2017Doctoral thesis

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Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF-κB activation

Publication . Simões, André; Pereira, D. M.; Gomes, S. E.; Brito, H.; Carvalho, Tânia; French, A.; Castro, Rui E.; Steer, C. J.; Thibodeau, S. N.; Rodrigues, Cecília M. P.; Borralho, P. M.

This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (P<0.01) and adenocarcinomas (P<0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (P<0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (P<0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (P<0.05) and increased cell migration (P<0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (P<0.05), together with increased expression of the mesenchymal marker vimentin (P<0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (P<0.05). Finally, in the mouse model, lymph node metastasis was exclusively seen in orthotopically implanted tumours with overactivated MEK5/ERK5, and not in tumours with inhibited MEK5/ERK5. Our results suggested that MEK5/ERK5/NF-κB signalling pathway is important for tumour onset, progression and metastasis, possibly representing a novel relevant therapeutic target in colon cancer treatment.

2015Journal article

Open access