Differential Effects of Trp53 Alterations in Murine Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) development is a multi-step process resulting in the accumulation of genetic alterations. Despite its high incidence, there are currently no mouse models that accurately recapitulate this process and mimic sporadic CRC. We aimed to develop and characterize a genetically engineered mouse model (GEMM) of Apc/Kras/Trp53 mutant CRC, the most frequent genetic subtype of CRC. METHODS: Tumors were induced in mice with conditional mutations or knockouts in Apc, Kras, and Trp53 by a segmental adeno-cre viral infection, monitored via colonoscopy and characterized on multiple levels via immunohistochemistry and next-generation sequencing. RESULTS: The model accurately recapitulates human colorectal carcinogenesis clinically, histologically and genetically. The Trp53 R172H hotspot mutation leads to significantly increased metastatic capacity. The effects of Trp53 alterations, as well as the response to treatment of this model, are similar to human CRC. Exome sequencing revealed spontaneous protein-modifying alterations in multiple CRC-related genes and oncogenic pathways, resulting in a genetic landscape resembling human CRC. CONCLUSIONS: This model realistically mimics human CRC in many aspects, allows new insights into the role of TP53 in CRC, enables highly predictive preclinical studies and demonstrates the value of GEMMs in current translational cancer research and drug development.

GAS2L1 Is a Potential Biomarker of Circulating Tumor Cells in Pancreatic Cancer.

Pancreatic cancer is a malignant disease with high mortality and a dismal prognosis. Circulating tumor cell (CTC) detection and characterization have emerged as essential techniques for early detection, prognostication, and liquid biopsy in many solid malignancies. Unfortunately, due to the low EPCAM expression in pancreatic cancer CTCs, no specific marker is available to identify and isolate this rare cell population. This study analyzed single-cell RNA sequencing profiles of pancreatic CTCs from a genetically engineered mouse model (GEMM) and pancreatic cancer patients. Through dimensionality reduction analysis, murine pancreatic CTCs were grouped into three clusters with different biological functions. CLIC4 and GAS2L1 were shown to be overexpressed in pancreatic CTCs in comparison with peripheral blood mononuclear cells (PBMCs). Further analyses of PBMCs and RNA-sequencing datasets of enriched pancreatic CTCs were used to validate the overexpression of GAS2L1 in pancreatic CTCs. A combinatorial approach using both GAS2L1 and EPCAM expression leads to an increased detection rate of CTCs in PDAC in both GEMM and patient samples. GAS2L1 is thus proposed as a novel biomarker of pancreatic cancer CTCs.

Characterization of Stem-like Circulating Tumor Cells in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most frequent cause of death from cancer. Circulating tumor cells (CTCs) with stem-like characteristics lead to distant metastases and thus contribute to the dismal prognosis of PDAC. Our purpose is to investigate the role of stemness in CTCs derived from a genetically engineered mouse model of PDAC and to further explore the potential molecular mechanisms. The publically available RNA sequencing dataset GSE51372 was analyzed, and CTCs with (CTC-S) or without (CTC-N) stem-like features were discriminated based on a principal component analysis (PCA). Differentially expressed genes, weighted gene co-expression network analysis (WGCNA), and further functional enrichment analyses were performed. The prognostic role of the candidate gene (CTNNB1) was assessed in a clinical PDAC patient cohort. Overexpression of the pluripotency marker Klf4 (Krüppel-like factor 4) in CTC-S cells positively correlates with Ctnnb1 (ß-Catenin) expression, and their interaction presumably happens via protein-protein binding in the nucleus. As a result, the adherens junction pathway is significantly enriched in CTC-S. Furthermore, the overexpression of Ctnnb1 is a negative prognostic factor for progression-free survival (PFS) and relapse-free survival (RFS) in human PDAC cohort. Overexpression of Ctnnb1 may thus promote the metastatic capabilities of CTCs with stem-like properties via adherens junctions in murine PDAC.

The overall potential of CD47 in cancer immunotherapy: with a focus on gastrointestinal tumors.

Introduction: CD47 is an anti-phagocytic ('don't eat me') signal overexpressed in many malignant diseases. It acts as myeloid immune checkpoint and thus has prognostic and therapeutic implications.Areas covered: This review presents and discusses the currently available data on the prognostic role and therapeutic value of CD47 in gastrointestinal tumors.Expert opinion: CD47 is overexpressed on the great majority of gastrointestinal tumors, cancer stem cells and circulating tumor cells. Overexpression of CD47 usually predicts a negative prognosis and seems to contribute to cancer immune evasion. The inhibition of CD47 has shown impressive results in clinical trials in hematologic malignancies. However, for gastrointestinal tumors only preclinical data is available. Inhibition of this myeloid immune checkpoint may yield great clinical benefit due to the abundance of myeloid effector cells. However, due to the ubiquitous expression of CD47 and the resulting antigen sink, vast amounts of antibody are required in order to reach therapeutic concentrations. QPCTL inhibitors blocking post-translational modification of CD47 protein may be a solution to this problem.

The prognostic role of circulating tumor cells in colorectal cancer.

Introduction: Metastasis is the main cause of cancer-associated death in colorectal cancer (CRC). The presence of circulating tumor cells (CTC) in the blood is associated with an increased risk of recurrence and poor prognosis. The clinical significance of CTCs as a novel biomarker has been extensively studied in the last decade. It has been shown that CTC detection applies to early cancer detection. The presence of CTCs is associated with metastatic spread and poor survival and is also useful as a marker for therapy response.Areas covered: We summarize the role of CTC in CRC, their clinical significance, current methods for CTC detection and challenges as well as future perspectives of CTC research.Expert commentary: The clinical significance of CTC in CRC patients is well established. Although insightful, the available marker-based approaches hampered our understanding of the CTCs and their biology, as such approaches do not take into account the heterogeneity of these cell populations. New technologies should expand the marker-based detection to multi biomarker-based approaches together with recent technological advances in microfluidics for single cell enrichment and analysis.

Prognostic value of DLGAP5 in colorectal cancer.

PURPOSE: DLG7 (disc large homolog 7) is a microtubule-associated protein encoded by DLGAP5 (DLG associated protein 5) gene and has an important role during spindle assembly. Spindle assembly deregulation is a well-known cause of genomic instability. The aim of this study was to investigate the influence of DLGAP5 expression on survival and to evaluate its potential use as a biomarker in colorectal cancer (CRC). METHODS: DLGAP5 expression was measured in the primary tumor and corresponding normal mucosa samples from 109 patients with CRC and correlated to clinical and pathological data. The results were validated in a second, publically available patient cohort. Molecular effects of DLG7/DLGAP5 in CRC were analyzed via functional assays in knockdown cell lines. RESULTS: DLGAP5 downregulation led to a significant reduction of the invasion and migration potential in CRC. In addition, DLGAP5 expression correlates with nodal status and advanced UICC stage (III-IV).Subgroup analyses revealed a correlation between DLGAP5 overexpression and poor survival in patients with non-metastatic disease (M0). Furthermore, overexpression of DLGAP5 is associated with worse overall survival in distinct molecular CRC subtypes. CONCLUSIONS: The results of this study suggest the importance of DLGAP5 in defining a more aggressive CRC phenotype. DLG7/DLGAP5 represents a potential biomarker for CRC in molecular subgroups of CRC.

Optical stimulation of cardiac cells with a polymer-supported silicon nanowire matrix.

Electronic pacemakers can treat electrical conduction disorders in hearts; however, they are invasive, bulky, and linked to increased incidence of infection at the tissue-device interface. Thus, researchers have looked to other more biocompatible methods for cardiac pacing or resynchronization, such as femtosecond infrared light pulsing, optogenetics, and polymer-based cardiac patches integrated with metal electrodes. Here we develop a biocompatible nongenetic approach for the optical modulation of cardiac cells and tissues. We demonstrate that a polymer-silicon nanowire composite mesh can be used to convert fast moving, low-radiance optical inputs into stimulatory signals in target cardiac cells. Our method allows for the stimulation of the cultured cardiomyocytes or ex vivo heart to beat at a higher target frequency.

Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.

Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1ß, IFN-γ; nitric oxide (·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O2·-), hydrogen peroxide (H2O2) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H2O2 generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1ß-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H2O2 to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H2O2 treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O2·- generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1ß-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.

Inositol 1, 4, 5-trisphosphate-dependent nuclear calcium signals regulate angiogenesis and cell motility in triple negative breast cancer.

Increases in nuclear calcium concentration generate specific biological outcomes that differ from those resulting from increased cytoplasmic calcium. Nuclear calcium effects on tumor cell proliferation are widely appreciated; nevertheless, its involvement in other steps of tumor progression is not well understood. Therefore, we evaluated whether nuclear calcium is essential in other additional stages of tumor progression, including key steps associated with the formation of the primary tumor or with the metastatic cascade. We found that nuclear calcium buffering impaired 4T1 triple negative breast cancer growth not just by decreasing tumor cell proliferation, but also by enhancing tumor necrosis. Moreover, nuclear calcium regulates tumor angiogenesis through a mechanism that involves the upregulation of the anti-angiogenic C-X-C motif chemokine 10 (CXCL10-IP10). In addition, nuclear calcium buffering regulates breast tumor cell motility, culminating in less cell invasion, likely due to enhanced vinculin expression, a focal adhesion structural protein. Together, our results show that nuclear calcium is essential for triple breast cancer angiogenesis and cell migration and can be considered as a promising strategic target for triple negative breast cancer therapy.

Cholesterol depletion impairs contractile machinery in neonatal rat cardiomyocytes.

Cholesterol regulates numerous cellular processes. Depleting its synthesis in skeletal myofibers induces vacuolization and contraction impairment. However, little is known about how cholesterol reduction affects cardiomyocyte behavior. Here, we deplete cholesterol by incubating neonatal cardiomyocytes with methyl-beta-cyclodextrin. Traction force microscopy shows that lowering cholesterol increases the rate of cell contraction and generates defects in cell relaxation. Cholesterol depletion also increases membrane tension, Ca2+ spikes frequency and intracellular Ca2+ concentration. These changes can be correlated with modifications in caveolin-3 and L-Type Ca2+ channel distributions across the sarcolemma. Channel regulation is also compromised since cAMP-dependent PKA activity is enhanced, increasing the probability of L-Type Ca2+ channel opening events. Immunofluorescence reveals that cholesterol depletion abrogates sarcomeric organization, changing spacing and alignment of α-actinin bands due to increase in proteolytic activity of calpain. We propose a mechanism in which cholesterol depletion triggers a signaling cascade, culminating with contraction impairment and myofibril disruption in cardiomyocytes.

Effects of cytoskeletal drugs on actin cortex elasticity.

Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.

Profiling of individual human red blood cells under osmotic stress using defocusing microscopy.

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities. We monitor the RBCs' radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed. Finally, membrane fluctuation measurements give us information about the cells deformability.

Trypasonoma cruzi uses a specific subset of host cell lysosomes for cell invasion.

Trypanosoma cruzi is an intracellular parasite that depends on host cell lysosome recruitment and fusion for cell infection. Recently, we have shown that host cells present two differentially regulated lysosome pools. Treatment with methyl-beta cyclodextrin, a drug able to sequester cholesterol from plasma membrane, triggers the exocytosis of peripheral lysosomes, while treatment with Latrunculin-A, an actin depolymerizing drug, recruits a more internal pool. In this work we aimed to study which pool is used by the T. cruzi during invasion. We have shown that invasion is impaired when cells are previously treated with methyl-beta cyclodextrin, but not with Latrunculin-A, indicating that T. cruzi uses the cortical pool for invasion.

Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MßCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MßCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MßCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MßCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MßCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.

Canine distemper virus induces apoptosis in cervical tumor derived cell lines.

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.