Theranostics Through the Synergistic Cooperation of Heterometallic Complexes

Heterometallic drugs are emerging as a great alternative to conventional metallodrugs. Careful selection of different metallic fragments makes possible to enhance not only the therapeutic potential by a synergistic effect, but also to incorpore key features like traceability. Drugs that integrate traceability and therapy in one system are known as theranostic agents. In cancer research, theranostic agents are becoming increasingly important. They deliver crucial information regarding their biological interplay that can ultimately be used for optimization. The well-established therapeutic potential of PtII-, RuII- and AuI-based drugs combined with the outstanding optical properties of d6 transition metal complexes grant the delivery of traceable metallodrugs. These species can be easily fine-tuned through modification of their respective ligands to provide a new generation of drugs

Luminescent Re(I)/Au(I) Species As Selective Anticancer Agents for HeLa Cells

A series of neutral and cationic heterotrimetallic complexes of the type fac-[Re(CO)3(bipy(CC)2-(AuL)2)X]n, where bipy(CC)2 is 4, 4'-alkynyl-2, 2'-bipyridine; L is either triphenylphosphine (PPh3), [1, 3-bis(2, 6-diisopropylphenyl)-imidazol-2-ylidene] (IPr), or tert-butyl isocyanide (CNtBu); and X is a chloride (n = 0) or acetonitrile (n = 1), were synthesized and characterized together with their Re(I) precursors, i.e., fac-[Re(CO)3(bipy(CC)2)X]n. X-ray diffraction of complexes 1, 3, and 6 corroborated the expected octahedral and linear distribution of the ligands along the Re(I) and Au(I) centers, respectively. Luminescent studies showed that all the complexes displayed a broad emission band centered between 565 and 680 nm, corresponding to a 3MLCT from the Re(I) to the diimine derivative. The presence of the gold fragment coordinated to the diimine ligand shifted in all cases the emission maxima toward higher energies. Such an emission difference could be potentially used for assessing the precise moment of interaction of the probe with the biological target if the gold fragment is implicated. Antiproliferative studies in cancer cells, A549 (lung cancer) and HeLa (cervix cancer), showed a generalized selectivity toward HeLa cells for those heterotrimetallic species incubated at longer times (72 vs 24 h). ICP-MS spectrometry revealed the greater cell internalization of cationic vs neutral species. Preliminary fluorescence microscopy experiments showed a different behavior of the complexes in HeLa and A549 cell lines. Whereas the complexes in A549 were randomly distributed in the outside of the cell, those incubated with HeLa cells were located close to the cellular membrane, suggesting some type of interaction, and possibly explaining their cellular selectivity when it comes to the antiproliferative activity displayed in the different cell lines

Multifunctional heterometallic Iriii-Aui probes as promising anticancer and antiangiogenic agents

A new class of emissive cyclometallated IrIII-AuI complexes with a bis(diphenylphosphino) methanide bridging ligand was successfully synthesised from the diphosphino complex [Ir(N^C)2(dppm)]+ (1). The different gold ancillary ligand, a triphenylphosphine (2), a chloride (3) or a thiocytosine (4) did not reveal any significant effect on the photophysical properties, which are mainly due to metal-to-ligand charge-transfer (3MLCT) transitions based on IrIII. However, the AuI fragment, along with the ancillary ligand, seemed crucial for the bioactivity in A549 lung carcinoma cells versus endothelial cells. Both cell types display variable sensitivities to the complexes (IC50=0.6–3.5 µM). The apoptotic pathway is activated in all cases, and paraptotic cell death seems to take place at initial stages in A549 cells. Species 2–4 showed at least dual lysosomal and mitochondrial biodistribution in A549 cells, with an initial lysosomal localisation and a possible trafficking process between both organelles with time. The bimetallic IrIII-AuI complexes disrupted the mitochondrial transmembrane potential in A549 cells and increased reactive oxygen species (ROS) generation and thioredoxin reductase (TrxR) inhibition in comparison with that displayed by the monometallic complex 1. Angiogenic activity assays performed in endothelial cells revealed the promising antimetastatic potential of 1, 2 and 4. © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH

Caracterización de exosomas obtenidos de líquido cefalorraquídeo de ovino con scrapie y desarrollo de modelos celulares de la enfermedad

Las encefalopatías espongiformes transmisibles (EETs) son un grupo de enfermedades neurodegenerativas letales que afectan a animales y humanos y se caracterizan por la acumulación de una isoforma patológica (PrPSc) de la proteína prión celular (PrPC) en las células nerviosas. Con el fin de identificar potenciales biomarcadores para el diagnóstico de las EETs, se analizó la expresión de una batería de miRNAs que se han visto alterados en distintas enfermedades neurodegenerativas en líquido cefalorraquídeo (LCR) de ovino con scrapie, el prototipo animal de las EETs. Se observaron diferencias significativas entre animales enfermos y sanos para los niveles de miR-486-5p, miR-146a-5p y miR-21-5p. Dado que los exosomas transportan entre células factores que favorecen el desarrollo de la patología, se realizaron los mismos análisis en la fracción exosomal del LCR, sin embargo, el aislamiento de exosomas no facilitó la detección de miRNAs desregulados dada su baja concentración. Por otro lado, se planteó implementar un modelo celular para el estudio de las EETs. Con este objetivo, se cultivaron, tanto en condiciones de crecimiento estándar como de diferenciación neurogénica, células madre mesenquimales (MSCs) procedentes de la médula ósea de 3 ovejas con genotipos relacionados con una alta susceptibilidad al scrapie (ARQ/ARQ, VRQ/VRQ y ARQ/VRQ). Las MSCs sometidas a diferenciación neurogénica sufrieron ligeros cambios morfológicos y el descenso de la expresión del gen que codifica el factor de transcripción OCT4, asociado a la pluripotencia de las células madre. Además se vio alterada la expresión de genes asociados a la replicación priónica. Después se inocularon los cultivos con extracto cerebral de ovino con scrapie y se analizó la cantidad de proteína patológica en los mismos a distintos tiempos. Los cultivos mostraron distinta capacidad de replicación del prión y la inoculación produjo alteraciones en la proliferación y la expresión génica de las MSCs. Aunque hay que seguir investigando para confirmar el efecto del genotipo en la respuesta a la infección, los resultados obtenidos confirman el potencial de las MSCs como modelo para el estudio de las EETs

Epigenetic alterations leading to TMPRSS4 promoter hypomethylation and protein overexpression predict poor prognosis in squamous lung cancer patients

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped

Identification of a novel synthetic lethal vulnerability in non-small cell lung cancer by co-targeting TMPRSS4 and DDR1

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.This work was supported by FIMA, Centro de Investigacion Biomedica en Red de Cancer (CIBERONC) (grant number: CB16/12/00443), Fundacion Cientifica Asociacion Espanola Contra el Cancer (grant number: GCB14-2170), Fundacion Ramon Areces, Instituto de Salud Carlos III and the European Regional Development Fund (ERDF, A way to make Europe) (grant numbers: PI19/00098; PI19/00230; PI20/00419), Fundacion Roberto Arnal Planelles and an IASLC Fellowship funding (K.V.); D.S. was supported by the Juan de la Cierva-Incorporacion program, Spanish Ministry of Science and Innovation (grant number: IJCI-2016-27595); E.R. was supported by a FPU, Spanish Ministry of Education ( grant number: FPU17/01168); M.E. was supported by PFIS, Spanish Ministry of Health, M.L. was supported by a Junior Investigator grant from AECC

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis

TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC

Identification of novel synthetic lethal vulnerability in non small cell lung cancer by co targeting TMPRSS4 and DDR1

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4