Inhibiting HER3 Hyperphosphorylation in HER2-Overexpressing Breast Cancer through Multimodal Therapy with Branched Gold Nanoshells.

Treatment failure in breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) is associated mainly to the upregulation of human epidermal growth factor receptor 3 (HER3) oncoprotein linked to chemoresitence. Therefore, to increase patient survival, here a multimodal theranostic nanoplatform targeting both HER2 and HER3 is developed. This consists of doxorubicin-loaded branched gold nanoshells functionalized with the near-infrared (NIR) fluorescent dye indocyanine green, a small interfering RNA (siRNA) against HER3, and the HER2-specific antibody Transtuzumab, able to provide a combined therapeutic outcome (chemo- and photothermal activities, RNA silencing, and immune response). In vitro assays in HER2+ /HER3+ SKBR-3 breast cancer cells have shown an effective silencing of HER3 by the released siRNA and an inhibition of HER2 oncoproteins provided by Trastuzumab, along with a decrease of the serine/threonine protein kinase Akt (p-AKT) typically associated with cell survival and proliferation, which helps to overcome doxorubicin chemoresistance. Conversely, adding the NIR light therapy, an increment in p-AKT concentration is observed, although HER2/HER3 inhibitions are maintained for 72 h. Finally, in vivo studies in a tumor-bearing mice model display a significant progressively decrease of the tumor volume after nanoparticle administration and subsequent NIR light irradiation, confirming the potential efficacy of the hybrid nanocarrier.

A New Opportunity for "Old" Molecules: Targeting PARP1 Activity through a Non-Enzymatic Mechanism.

In recent years, new therapies have been developed based on molecules that target molecular mechanisms involved in both the initiation and maintenance of the oncogenic process. Among these molecules are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. PARP1 has emerged as a target with great therapeutic potential for some tumor types, drawing attention to this enzyme and resulting in many small molecule inhibitors of its enzymatic activity. Therefore, many PARP inhibitors are currently in clinical trials for the treatment of homologous recombination (HR)-deficient tumors, BRCA-related cancers, taking advantage of synthetic lethality. In addition, several novel cellular functions unrelated to its role in DNA repair have been described, including post-translational modification of transcription factors, or acting through protein-protein interactions as a co-activator or co-repressor of transcription. Previously, we reported that this enzyme may play a key role as a transcriptional co-activator of an important component of cell cycle regulation, the transcription factor E2F1. Here, we show that PARP inhibitors, which interfere with its activity in cell cycle regulation, perform this without affecting its enzymatic function.

Development of a biosensor based on a new marine luciferase fused to an affibody to assess Her2 expression in living cells.

The development of new diagnostic tools in tumor pathology allows the optimization of individualized therapies in cancer patients. The functional optical image provides a unique opportunity to identify the pathophysiological characteristics of each tumor in a non-invasive way. Although fluorescent recombinant affibodies and nanobodies, capable of detecting certain membrane proteins present in tumor cells, has been described, the use of bioluminescent molecules is gaining a great impact in this field due to its high sensitivity. In this work, we characterize a new luciferase from the Metridia lucens copepod (MlLuc) and develop a novel bioluminescent recombinant affibody (MlLuc-aff) capable of recognizing the HER2 receptors that are overexpressed in breast cancer tumors. For this purpose, the thermostability and pH sensitivity of MlLuc1.1 were determined, showing no significant changes in the activity among temperatures between 4 and 70 °C, and with a maximum of brightness at pH 8.0. Furthermore, MlLuc-aff was able to accurately detect HER2 receptors expressed in the SK-BR-3 cells. Future applications of this new tracer can contribute to the early diagnosis of breast cancer patients and the assessment of the efficacy of the treatment.

Development of a nanocapsule-loaded hydrogel for drug delivery for intraperitoneal administration.

Intraperitoneal (IP) drug delivery of chemotherapeutic agents, administered through hyperthermal intraperitoneal chemotherapy (HIPEC) and pressurized intraperitoneal aerosolized chemotherapy (PIPAC), is effective for the treatment of peritoneal malignancies. However, these therapeutic interventions are cumbersome in terms of surgical practice and are often associated with the formation of peritoneal adhesions, due to the catheters inserted into the peritoneal cavity during these procedures. Hence, there is a need for the development of drug delivery systems that can be administered into the peritoneal cavity. In this study, we have developed a nanocapsule (NCs)-loaded hydrogel for drug delivery in the peritoneal cavity. The hydrogel has been developed using poly(ethylene glycol) (PEG) and thiol-maleimide chemistry. NCs-loaded hydrogels were characterized by rheology and their resistance to dilution and drug release were determined in vitro. Using IVIS® to measure individual organ and recovered gel fluorescence intensity, an in vivo imaging study was performed and demonstrated that NCs incorporated in the PEG gel were retained in the IP cavity for 24 h after IP administration. NCs-loaded PEG gels could find potential applications as biodegradable, drug delivery systems that could be implanted in the IP cavity, for example at a the tumour resection site to prevent recurrence of microscopic tumours.

From neural stem cells to glioblastoma: A natural history of GBM recapitulated in vitro.

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.

PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer.

In recent years, poly (ADP-ribose) polymerase (PARP) inhibitors have been evaluated for treating homologous recombination-deficient tumours, taking advantage of synthetic lethality. However, increasing evidence indicates that PARP1 exert several cellular functions unrelated with their role on DNA repair, including function as a co-activator of transcription through protein-protein interaction with E2F1. Since the RB/E2F1 pathway is among the most frequently mutated in many tumour types, we investigated whether the absence of PARP activity could counteract the consequences of E2F1 hyperactivation. Our results demonstrate that genetic ablation of Parp1 extends the survival of Rb-null embryos, while genetic inactivation of Parp1 results in reduced development of pRb-dependent tumours. Our results demonstrate that PARP1 plays a key role as a transcriptional co-activator of the transcription factor E2F1, an important component of the cell cycle regulation. Considering that most oncogenic processes are associated with cell cycle deregulation, the disruption of this PARP1-E2F1 interaction could provide a new therapeutic target of great interest and a wide spectrum of indications.

Immortalization of a cell line with neural stem cell characteristics derived from mouse embryo brain.

BACKGROUND: Neural stem cells (NSC) have been extensively used as a tool to investigate the mechanisms responsible for neural repair, and they have been also considered as the source for a series of promising replacement therapies in various neurodegenerative diseases. However, their use is limited by their relative rarity and anatomical localization, and also because, the methods for isolation and characterization are usually time consuming and have some technical limitations. RESULTS: In this study, we describe a resource and method for obtaining immortalized cells with NSC characteristics obtained from mouse brain embryo. CONCLUSIONS: Because these cells can be maintained indefinitely in culture, they may constitute a permanent source of NSC that can be used for research studies on neural development and regeneration.

A Bio-inspired Hypoxia Sensor using HIF1a-Oxygen-Dependent Degradation Domain.

Functional imaging has become an important tool in oncology because it not only provides information about the size and localization of the tumour, but also about the pathophysiological features of the tumoural cells. One of the characteristic features of some tumour types is that their fast growth leads to deficient intratumoral vascularization, which results in low oxygen availability. To overcome this lack of oxygen, tumoural cells activate the neoangiogenic program by upregulating the transcription factor HIF-1α. Herein we report a non-invasive in vitro detection method of hypoxia using designed fluorescent peptide probes based on the oxygen-dependent degradation domain of HIF-1α. The fluorescent probe retains the oxygen-sensing capability of HIF-1α, so that it is stabilized under hypoxia and readily degraded by the proteasome under normoxia, thus providing direct information of the cellular oxygen availability.

Expression of myostatin in human hematopoietic cells unveils novel autocrine/paracrine actions for the hormone.

Myostatin is a member of the transforming growth factor ß (TGFß) superfamily that has a well-established role as a mediator of muscle growth and development. However, myostatin is now emerging as a pleiotropic hormone with multiple actions in the regulation of the metabolism as well as several aspects of both cardiac and smooth muscle cells physiology. In addition, myostatin is also expressed in several nonmuscular cells where its physiological role remains to be elucidated in most cases. In this report, we have shown that both myostatin and its receptor system are expressed in blood cells and in hematopoietic cell lines. Furthermore, myostatin treatment promotes differentiation of both HL60 and K562 cells through a mechanism that involves activation of extracellular signal-regulated kinases 1/2 and p38-mitogen-activated protein kinase, thus leading to the possibility that myostatin may be a paracrine/autocrine factor involved in the control of haematopoiesis. In addition, the presence of myostatin expression in immune cells could envisage a novel role for the hormone in the pathogenesis of inflammatory diseases.

NK Cell-Based Glioblastoma Immunotherapy.

Glioblastoma (GB) is the most aggressive and most common malignant primary brain tumor diagnosed in adults. GB shows a poor prognosis and, unfortunately, current therapies are unable to improve its clinical outcome, imposing the need for innovative therapeutic approaches. The main reason for the poor prognosis is the great cell heterogeneity of the tumor mass and its high capacity for invading healthy tissues. Moreover, the glioblastoma microenvironment is capable of suppressing the action of the immune system through several mechanisms such as recruitment of cell modulators. Development of new therapies that avoid this immune evasion could improve the response to the current treatments for this pathology. Natural Killer (NK) cells are cellular components of the immune system more difficult to deceive by tumor cells and with greater cytotoxic activity. Their use in immunotherapy gains strength because they are a less toxic alternative to existing therapy, but the current research focuses on mimicking the NK attack strategy. Here, we summarize the most recent studies regarding molecular mechanisms involved in the GB and immune cells interaction and highlight the relevance of NK cells in the new therapeutic challenges.

Uncoupling Oncogene-Induced Senescence (OIS) and DNA Damage Response (DDR) triggered by DNA hyper-replication: lessons from primary mouse embryo astrocytes (MEA).

Oncogene-induced senescence (OIS) is a complex process, in which activation of oncogenic signals during early tumorigenesis results in a high degree of DNA replication stress. The ensuing response to the DNA damage produces a permanent G1 arrest that prevents unlimited cell proliferation and lessens the development of tumours. However, despite the role of OIS in the proliferative arrest resulting from an activating oncogenic-lesion has obtained wide support, there is also evidence indicating that cells may overcome oncogene-induced senescence under some circumstances. In this study, we have investigated the possibility that some of the assumptions on the role of DNA damage response (DDR) in triggering OIS may depend on the fact that most of the available data were obtained in mouse embryo fibroblast. By comparing the degree of OIS observed in mouse embryo fibroblasts (MEF) and mouse embryo astrocytes (MEA) obtained from the same individuals we have demonstrated that, despite truthful activation of DDR in both cell types, significant levels of OIS were only detected in MEF. Therefore, this uncoupling between OIS and DDR observed in astrocytes supports the intriguingly possibility that OIS is not a widespread response mechanism to DDR.

RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αßγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

Varias teorías pretenden explicar la transformación maligna de las células, como es la mutación de genes supresores de tumor y proto-oncogenes. La deleción de Rb (un supresor de tumor), la sobreexpresión de Ras mutado (un proto-oncogén), o ambos, son suficientes para desarrollar gliomagénesis in vitro, y estas características genéticas se asocian con su alta tasa de proliferación. Un rasgo distintivo del cáncer es la capacidad de las células tumorales para evadir el sistema inmune. Por lo que en este estudio analizamos si las mutaciones específicas están relacionadas con la evasión de la respuesta inmune. Para abordar esta cuestión, tres líneas celulares de glioma transformadas se obtuvieron (Rb−/−, RasV12, y Rb−/−/RasV12) mediante transformación retroviral de astrocitos in vitro, reportado anteriormente. Además, las células transformadas RasV12 y Rb−/−/RasV12 fueron inyectadas en ratones SCID y después del crecimiento del tumor se obtuvieron dos líneas celulares de glioma estables. En todas las células se determinaron la expresión génica Rb y Ras, morfología, capacidad de proliferación, expresión de MHC I, Rae1δ, and Rae1αßγδε, mult1, H60a, H60b, H60c, como ligandos para receptores de células NK, y su susceptibilidad a la citotoxicidad mediada por células NK. Nuestros resultados muestran que la transformación de los astrocitos (pérdida de Rb, la sobreexpresión de Ras, o ambos) indujo cambios fenotípicos y funcionales asociados con la resistencia a la citotoxicidad mediada por células NK. Además, la transferencia de astrocitos transformados dentro de ratones SCID aumento la resistencia a la citotoxicidad mediada por células NK, lo que se sugiere que los cambios específicos en un supresor de tumores (Rb) y un proto-oncogén (Ras) son suficientes para conferir resistencia a la citotoxicidad mediada por células NK en células de glioma y, por tanto, proporcionar una idea de la capacidad de las células tumorales para evadir la respuesta inmune.

The antimitotic potential of PARP inhibitors, an unexplored therapeutic alternative.

ADP-ribosylation or PARsylation is one of the most abundant modifications of proteins and DNA. Although the usual context for PARsylation involves the detection and repair of DNA damage in the cell, poly(ADP-ribose) polymerases are known to regulate a number of biological processes besides maintaining genome integrity. One of these processes is the assembly and maintenance of the mitotic spindle where the presence of PARP-1 and tankyrase 1 (TNKS1), two of the best-characterized members of the PARP superfamily, is of critical importance. Here, we recapitulate the biological implications of the absence of poly(ADP-ribose) polymerases and depletion of PARsylation occurrence in mitosis in order to better understand the antimitotic effects of PARP inhibitors. In this regard, we also present an overview of the existing and more relevant molecules, with a special attention to the historical development of their pharmacological properties and structures, as well as a brief summary of clinical trials involving PARP inhibitors.

Fluorescent drug-loaded, polymeric-based, branched gold nanoshells for localized multimodal therapy and imaging of tumoral cells.

Here we report the synthesis of PLGA/DOXO-core Au-branched shell nanostructures (BGNSHs) functionalized with a human serum albumin/indocyanine green/folic acid complex (HSA-ICG-FA) to configure a multifunctional nanotheranostic platform. First, branched gold nanoshells (BGNSHs) were obtained through a seeded-growth surfactant-less method. These BGNSHs were loaded during the synthetic process with the chemotherapeutic drug doxorubicin, a DNA intercalating agent and topoisomerase II inhibitior. In parallel, the fluorescent near-infrared (NIR) dye indocyanine green (ICG) was conjugated to the protein human serum albumin (HSA) by electrostatic and hydrophobic interactions. Subsequently, folic acid was covalently attached to the HSA-ICG complex. In this way, we created a protein complex with targeting specificity and fluorescent imaging capability. The resulting HSA-ICG-FA complex was adsorbed to the gold nanostructures surface (BGNSH-HSA-ICG-FA) in a straightforward incubation process thanks to the high affinity of HSA to gold surface. In this manner, BGNSH-HSA-ICG-FA platforms were featured with multifunctional abilities: the possibility of fluorescence imaging for diagnosis and therapy monitoring by exploiting the inherent fluorescence of the dye, and a multimodal therapy approach consisting of the simultaneous combination of chemotherapy, provided by the loaded drug, and the potential cytotoxic effect of photodynamic and photothermal therapies provided by the dye and the gold nanolayer of the hybrid structure, respectively, upon NIR light irradiation of suitable wavelength. The combination of this trimodal approach was observed to exert a synergistic effect on the cytotoxicity of tumoral cells in vitro. Furthermore, FA was proved to enhance the internalization of nanoplatform. The ability of the nanoplatforms as fluorescence imaging contrast agents was tested by preliminary analyzing their biodistribution in vivo in a tumor-bearing mice model.

AMPK activation by oncogenesis is required to maintain cancer cell proliferation in astrocytic tumors.

5'-AMP-activated protein kinase (AMPK) is an energy sensor that controls cell metabolism, and it has been related to apoptosis and cell-cycle arrest. Although its role in metabolic homeostasis is well documented, its function in cancer is much less clear. In this study, we examined the role of AMPK in a mouse model of astrocytoma driven by oncogenic H-Ras(V12) and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We also evaluated the activity and role of AMPK in human glioblastoma cells and xenografts. AMPK was constitutively activated in astrocytes expressing oncogenic H-Ras(V12) in parallel with high cell division rates. Genetic deletion of AMPK or attenuation of its activity in these cells was sufficient to reduce cell proliferation. The levels of pAMK were always related to the levels of phosphorylated retinoblastoma (Rb) at Ser804, which may indicate an AMPK-mediated phosphorylation of Rb. We confirmed this AMPK-Rb relationship in human glioblastoma cell lines and xenografts. In clinical specimens of human glioblastoma, elevated levels of activated AMPK appeared especially in areas of high proliferation surrounding the blood vessels. Together, our findings indicate that the initiation and progression of astrocytic tumors relies upon AMPK-dependent control of the cell cycle, thereby identifying AMPK as a candidate therapeutic target in this setting.

Defining hypoxic microenvironments by non-invasive functional optical imaging.

Functional imaging has become an important tool in oncology by informing about localisation and size of the tumour as well as the pathophysiological features of tumoural cells. One of the most characteristic features of some tumour types is the activation of the neoangiogenic programme which is specifically mediated by the transcription factor hypoxia-inducible factor (HIF)-1α, an important player in regulating this process and a prognostic marker of tumoural aggressiveness. Here we report a non-invasive in vivo detection of lung micrometastases in a mouse model of breast cancer using self-illuminating genetically encoded tracers responsive to intracellular HIF-1α levels and a preliminary analysis of the contribution of the tumoural masses to the metastatic niche. This model lays the foundations for novel hypoxia sensing probes able to detect micrometastatic disease with high sensitivity and specificity. Thus, optical functional imaging shows promise in the understanding of disease, drug development or image-guided therapy.