Isolda Casanova

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

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Codon 12 and codon 13 mutations at the K-ras gene induce different soft tissue sarcoma types in nude mice

K‐ras codon 12 mutation is more oncogenic in in vitro and in vivo experimental systems than Kras codon 13 mutation. Moreover, human colorectal tumors bearing a codon 12 mutation are more aggressive, invasive, and metastatic than the same tumor types carrying a codon 13 mutation. However, despite the association between specific sarcoma types and codon 12 or codon 13 mutations, the relationship between the position of the mutated codon at ras genes and tumor aggressiveness has not been studied in this tumor type. Here, we used a nude mice model to evaluate the tumorogenic capacity of stable transfectants of NIH3T3 fibroblasts, expressing Kras mutated at codon 12 (K12) or 13 (K13), and morphologically, functionally, and molecularly compared these tumors. We found histopathological differences between them, K12‐derived tumors showing fibrosarcoma‐like features, whereas K13‐derived tumors resembled malignant fibrous histiocytomas. Moreover, K12 tumors showed shorter latency of appearance, lower apoptotic and mitotic rates, and higher expression of markers for sarcoma aggressiveness (Ki67, p53 and c‐myc) than K13 tumors. They also showed differences in the expression or activation of Ras, Ras downstream pathways [c‐Jun N‐terminal kinase (JNK), MAPK and AKT], and apoptotic [AKT, Bcl‐2, Focal adhesion kinase (FAK)] and mitotic (cyclin B1) regulators, which could explain their functional differences. Most remarkably, the significantly diminished apoptotic rate observed in K12‐derived tumors was associated with enhanced antiapoptotic signaling through the AKT pathway. These morphological, functional, and molecular differences demonstrate that codon 12 and codon 13 mutations in the K‐ras oncogene can induce two different soft tissue sarcoma types in our in vivo model.

Ku70 predicts response and primary tumor recurrence after therapy in locally advanced head and neck cancer

5‐Fluorouracil and cisplatin‐based induction chemotherapy (IC) is commonly used to treat locally advanced head and neck squamous cell carcinoma (HNSCC). The role of nonhomologous end joining (NHEJ) genes (Ku70, Ku80 and DNA‐PKcs) in double‐strand break (DSB) repair, genomic instability and apoptosis suggest a possible impact on tumor response to radiotherapy, 5‐fluorouracil or cisplatin, as these agents are direct or indirect inductors of DSBs. We evaluated the relationship between Ku80, Ku70 or DNA PKcs mRNA expression in pretreatment tumor biopsies, and tumor response to IC or local recurrence, in 50 patients with HNSCC. Additionally, in an independent cohort of 75 patients with HNSCC, we evaluated the relationship between tumor Ku70 protein expression and the same clinical outcomes or patient survival. Tumors in the responder group had significantly higher mRNA levels for Ku70, Ku80 and DNA‐PKcs than those in the nonresponder group. Ku70 mRNA was the marker most significantly associated with response to IC. Moreover, high tumor Ku70 mRNA expression was associated with significantly longer local recurrence‐free survival (LRFS). Ku70 protein expression was also significantly related to response, and patients with higher percentage of tumor cells expressing Ku70 had longer LRFS. In addition, the percentage of Ku70 positive cells, tumor localization and node involvement were significantly associated with overall survival of patient. Therefore, Ku70 expression is a candidate predictive marker that could distinguish patients who are likely to benefit from chemoradiotherapy or radiotherapy after the induction chemotherapy treatment, suggesting a contribution of the NHEJ system in HNSCC clinical outcome.

Celecoxib induces anoikis in human colon carcinoma cells associated with the deregulation of focal adhesions and nuclear translocation of p130Cas

Celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, is effective as chemopreventive against colon cancer and it is the only nonsteoroidal antiinflammatory drug approved by the FDA for adjuvant therapy in patients with familial adenomatous polyposis. It is also being evaluated, within Phase II and III clinical trials, in combination with standard chemotherapy to treat sporadic colorectal cancer. Nevertheless, its antitumor mechanism of action is still not fully understood. In this study, we have evaluated the in vitro growth inhibitory effect of celecoxib in colon carcinoma cells and analyzed its mechanism of action. We report that the deregulation of the focal adhesion assembly protein Crk‐associated substrate 130 kDa (p130Cas) by celecoxib plays a relevant role in the cytotoxic effect of this drug. Thus, celecoxib induces the proteolysis of p130Cas and the nuclear translocation of the 31 kDa generated fragment leading to apoptosis. Furthermore, overexpression of wild‐type p130Cas reverts, in part, the growth inhibitory effect of celecoxib. In contrast, FAK and AKT do not appear to be involved in this activity. Our data suggest, for the first time, that the antitumor mechanism of action of celecoxib includes the induction of anoikis, an effect that is not related to COX‐2 inhibition. Besides providing new insights into the antitumor effect of celecoxib, this novel mechanism of action holds potential relevance in drug development. Indeed, our results open the possibility to develop new celecoxib derivatives that induce anoikis without COX‐2 inhibition so as to avoid the cardiovascular toxicity recently described for the COX‐2 inhibitors.

CXCR4 expression enhances diffuse large B cell lymphoma dissemination and decreases patient survival.

The chemokine receptor CXCR4 has been implicated in the migration and trafficking of malignant B cells in several haematological malignancies. Over‐expression of CXCR4 has been identified in haematological tumours, but data concerning the role of this receptor in diffuse large B cell lymphoma (DLBCL) are lacking. CXCR4 is a marker of poor prognosis in various neoplasms, correlating with metastatic disease and decreased survival of patients. We studied CXCR4 involvement in cell migration in vitro and dissemination in vivo. We also evaluated the prognostic significance of CXCR4 in 94 biopsies of DLBCL patients. We observed that the level of expression of CXCR4 in DLBCL cell lines correlated positively with in vitro migration. Expression of the receptor was also associated with increased engraftment and dissemination, and decreased survival time in NOD/SCID mice. Furthermore, administration of a specific CXCR4 antagonist, AMD3100, decreased dissemination of DLBCL cells in a xenograft mouse model. In addition, we found that CXCR4 expression is an independent prognostic factor for shorter overall survival and progression‐free survival in DLBCL patients. These results show that CXCR4 mediates dissemination of DLBCL cells and define for the first time its value as an independent prognostic marker in DLBCL patients. Copyright

Site-Dependent E-Cadherin Cleavage and Nuclear Translocation in a Metastatic Colorectal Cancer Model

Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1(OE)) or E-cadherin (E-cadherin(OE)), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, β-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1(OE) cells disseminated only to lymph nodes, whereas Mock or E-cadherin(OE) cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherin(OE) cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin(OE) cells overexpressed E-cadherin, but they did not show E-cadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment.

A celecoxib derivative inhibits focal adhesion signaling and induces caspase-8-dependent apoptosis in human acute myeloid leukemia cells

Most acute myeloid leukemias (AMLs), including those with c‐Kit or FLT3 mutations, show enhanced anchorage independent growth associated with constitutive activation of focal adhesion proteins. Moreover, these alterations increase cell survival, inhibit apoptosis and are associated with poor prognosis and resistance to chemotherapy. Therefore, the induction of apoptosis by selective inhibition of focal adhesion signaling may represent a novel anti‐AML therapy. Here, we have evaluated the antitumor effect and the mechanism of action of celecoxib and E7123, a non‐Cox‐2 inhibitor derivative, in a panel of human AML cell lines and bone marrow mononuclear cells from AML patients. Both compounds induce cell death by inhibiting focal adhesion signaling through p130Cas, FAK and c‐Src, leading to caspase‐8 dependent apoptosis. This mechanism of action differs from that of classical cytotoxic drugs or of other targeted therapies, and is amenable to rational drug development. Therefore, both drugs could be developed as AML therapeutics; nevertheless, E7123 shows more activity than celecoxib against AML cells, and may not present its Cox‐2 dependent cardiovascular toxicity. Finally, our results support the evaluation of celecoxib in AML patients, and the preclinical evaluation of E7123, before its possible clinical testing.

Gene expression signatures and molecular markers associated with clinical outcome in locally advanced head and neck carcinoma

The purpose of this study was to identify molecular markers associated with tumor recurrence and survival in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). We studied the expression profile of 63 pre-treatment tumor biopsies obtained from locally advanced HNSCCs treated with standard treatments. Cluster analysis identified three tumor subtypes associated with significant differences in local recurrence-free survival (LRFS) (P<0.001), progression free-survival (PFS) (P<0.009) and overall survival (OS) (P<0.004). Tumor subtype 1, associated with short LRFS, PFS and OS, showed features of epithelial-mesenchymal transition and undifferentiation. It also overexpressed genes involved in cell adhesion, NF-κB and integrin signalling. Tumor subtype 3, associated with longer LRFS, PFS and OS, showed a high degree of differentiation and overexpressed genes located in chromosomal regions 19q13 and 1q21. Tumor subtype 2, which had an intermediate clinical outcome between subtype 1 and subtype 3, overexpressed genes involved in branching morphogenesis. Finally, we validated the association between gene cluster classification and patient survival using Gene Set Enrichment Analysis and two HNSCC data sets obtained from two independent patient cohorts. In conclusion, we generated a gene prognostic signature associated with survival in locally advanced patients using the expression profile of the pre-treatment tumor biopsy. Independent prospective studies would be necessary to assess if the proposed survival signature could help to guide clinical management of HNSCC.

uPA/uPAR and SERPINE1 in head and neck cancer: role in tumor resistance, metastasis, prognosis and therapy

There is strong evidence supporting the role of the plasminogen activator system in head and neck squamous cell carcinoma (HNSCC), particularly of its uPA (urokinase plasminogen activator) / uPAR (urokinase plasminogen activator receptor) and SERPINE1 components. Overexpression of uPA/uPAR and SERPINE1 enhances tumor cell migration and invasion and plays a key role in metastasis development, conferring poor prognosis. The apparent paradox of uPA/uPAR and its inhibitor SERPINE1 producing similar effects is solved by the identification of SERPINE1 activated signaling pathways independent of uPA inhibition. Both uPA/uPAR and SERPINE1 are directly linked to the induction of epithelial-to-mesenchymal transition, the acquisition of stem cell properties and resistance to antitumor agents. The aim of this review is to provide insight on the deregulation of these proteins in all these processes. We also summarize their potential value as prognostic biomarkers or potential drug targets in HNSCC patients. Concomitant overexpression of uPA/uPAR and SERPINE1 is associated with a higher risk of metastasis and could be used to identify patients that would benefit from an adjuvant treatment. In the future, the specific inhibitors of uPA/uPAR and SERPINE1, which are still under development, could be used to design new therapeutic strategies in HNSCCs.