Biology subjects

Fernandez, A.

Publications and source records attributed to Fernandez, A..

61 records · Page 4Linked to original sources

bioRxiv2019

Phenylethynylbenzyl-Modified Biguanides Inhibit Pancreatic Cancer Tumor Growth

We present the design and synthesis of a small library of substituted biguanidium salts and their capacity to inhibit the growth/viability of pancreatic cancer cells. We first present their in vitro and membrane activity, before we address their mechanism of action in living cells and in vivo activity. We show that phenylethynyl biguanidium salts possess higher ability to cross hydrophobic barriers, improved mitochondrial accumulation and anticancer activity. Mechanistically, the most active compound 1b, like metformin, decreases the NAD+/NADH ratio and mitochondrial respiration, but at 800-fold lower concentration. In vivo studies with, the most active, compound 1b show a significant growth inhibition of pancreatic cancer xenografts in mice, while biguanides currently in clinical trials had no activity.

cancer biology↗

bioRxiv2018

Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr187, which was previously unreported. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was sufficient for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.\n\nSummary blurbMyc antagonizes p27 activity in cancer. Myc activates Cdk1 to phosphorylate p27, marking p27 for degradation. This depends on Myc-mediated cyclin A and B induction. Cdk1 inhibition is sufficient for a synthetic lethal interaction with Myc

cancer biology↗

bioRxiv2018

CCN4 shifts melanoma cells from a fragile proliferative state to a resilient metastatic state

While deregulated intracellular signaling initiates melanoma, intercellular crosstalk within the tumor microenvironment, often coordinated by soluble factors, is essential for melanoma progression and metastasis. One such secreted matricellular protein, cellular communication network factor 4 (CCN4), stimulates metastasis in other malignancies. Here, we report that CCN4 expression is associated progressively with reduced overall survival in patients with primary melanomas. To reveal the roles of CCN4 in melanoma progression, we used mouse melanoma models and knocked out Ccn4 using a homology-directed repair CRISPR/CAS9 system to generate pools of Ccn4-knockout cells. In vitro assays supported previous findings using clones generated using a double nickase-based CRISPR/CAS9 system that CCN4 promoted an epithelial - mesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. We also found that, while Ccn4 knockout enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions in vitro, quantitative analysis of tumor growth assays in vivo, and transcriptomics analysis of human melanoma cell lines suggested that CCN4 repressed cell growth and simultaneously enhanced cell survival. The collective role of CCN4 suggests a potential therapeutic target for limiting metastatic invasion in melanoma and a biomarker for metastatic potential.

cancer biology↗

bioRxiv2018

WNT1 Inducible Signaling Pathway Protein 1 (WISP1) Stimulates Melanoma Cell Invasion and Metastasis by Promoting Epithelial - Mesenchymal Transition (EMT)

Besides intrinsic changes, malignant cells release soluble signals to reshape their microenvironment. Among the signaling factors is WNT1 inducible signaling pathway protein 1 (WISP1), a secreted matricellular protein that is elevated in a variety of cancers including melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. In this work, we found that WISP1 knockout both increased cell proliferation and repressed wound healing, migration and invasion of mouse and human melanoma cells in an ensemble of in vitro assays. In vivo metastasis assays showed that WISP1 knockout repressed tumor metastasis in both C57BL/6Ncrl and NOD-scid IL2Rgammanull (NSG) mice with B16F10 and YUMM1.7 melanoma cells. Mechanistically, B16F10 cells that invaded in a transwell assay possessed a gene expression signature similar to Epithelial - Mesenchymal Transition (EMT), including coincident repression of E-cadherin and induction of fibronectin and N-cadherin. Upon WISP1 knockout, these EMT signature genes went in opposite directions in both mouse and human cell lines and were rescued by media containing WISP1 or recombinant WISP1 protein. In vivo, metastasis repression by WISP1 knockout was reversed by the reintroduction of either WISP1 or SNAI1. A set of EMT gene activation and inhibition experiments using recombinant WISP1 or kinase inhibitors in B16F10 and YUMM1.7 cells suggested that WISP1 activates Akt and MAP kinase signaling pathways to shift melanoma cells from a proliferative to invasive phenotype. Collectively, the results supported a model that WISP1 within the tumor microenvironment stimulates melanoma invasion and metastasis by promoting an EMT-like process.

cancer biology↗

bioRxiv2018

Bradycardic mice undergo effective heart rate improvement after specific homing to the sino-atrial node and differentiation of adult muscle derived stem cells.

Current treatments for heart automaticity disorders still lack a safe and efficient source of stem cells to restore normal biological pacemaking. Since adult Muscle-Derived Stem Cells (MDSC) show multi-lineage differentiation in vitro including into spontaneously beating cardiomyocytes, we questioned whether they could effectively differentiate into cardiac pacemakers, a specific population of cardiomyocytes producing electrical impulses in the sino-atrial node (SAN) of adult heart. We show here that beating cardiomyocytes, differentiated from MDSC in vitro, exhibit typical characteristics of cardiac pacemakers: expression of markers of the SAN lineage Hcn4, Tbx3 and Islet1, as well as spontaneous calcium transients and hyperpolarization-activated "funny" current and L-type Cav1.3 channels. Pacemaker-like myocytes differentiated in vitro from Cav1.3-deficient mouse stem cells produced slower rate of spontaneous Ca2+ transients, consistent with the reduced activity of native pacemakers in mutant mice. In vivo, undifferentiated wild type MDSC migrated and homed with increased engraftment to the SAN of bradycardic mutant Cav1.3-/- within 2-3 days after systemic I.P. injection. The increased homing of MDSCs corresponded to increased levels of the chemokine SDF1 and its receptor CXCR4 in mutant SAN tissue and was ensued by differentiation of MDSCs into Cav1.3-expressing pacemaker-like myocytes within 10 days and a significant improvement of the heart rate maintained for up to 40 days. Optical mapping and immunofluorescence analyses performed after 40 days on SAN tissue from transplanted wild type and mutant mice showed MDSCs integrated as pacemaking cells both electrically and functionally within recipient mouse SAN. These findings identify MDSCs as directly transplantable stem cells that efficiently home, differentiate and improve heart rhythm in mouse models of congenital bradycardia.

cell biology↗

bioRxiv2018

Senescent CD4 T cells unequivocally identify primary resistance and risk of hyperprogression to PD-L1/PD-1 immune checkpoint blockade in lung cancer

The majority of lung cancer patients are refractory to PD-L1/PD-1 blockade monotherapy. This therapy may even accelerate progression and death in a group of patients called hyperprogressors. Here we demonstrate that the efficacy of PD-L1/PD-1 blockade therapy relies on baseline circulating highly-differentiated CD28- CD27- CD4 T cells (THD cells), which segregate patients in two non-overlapping groups. THD cells in cancer patients mostly comprised of central memory subsets that potently co-upregulated PD-1 and LAG3 upon antigen recognition. Low baseline THD numbers unequivocally identified intrinsic non-responders and hyperprogressors, whom aberrantly responded to therapy with a potent systemic proliferative THD cell burst. Responder patients showed significant reductions in systemic CD4 THD cells throughout therapy linked to expansion of the CD28+ CD27+ CD4 T cell compartment. Quantification of THD cells from peripheral blood samples prior to therapy allows identification of non-responders, hyperprogressors and responders, a critical issue in clinical oncology. These results place CD4 T cell responses at the center of anti-tumor immunity.

cancer biology↗

bioRxiv2017

Non-canonical aberrant DNA hypermethylation in glioma

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC in primary samples from 54 gliomas and 72 colorectal cancer patients. Levels of 5hmC in colorectal cancer were very low and no consistent changes were detected between control tissues and tumors. As expected, levels of 5hmC in non-tumoral brain samples were high and significantly reduced at the 49,601 CpG sites in gliomas. Strikingly, hypo-hydroxymethylation at 4,627 (9.3%) of these CpG sites was associated with aberrant DNA hypermethylation. The DNA regions containing these CpG sites were enriched in H3K4me2, and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. We conclude that this data identifies a novel 5hmC-dependent non-canonical class of aberrant DNA hypermethylation in glioma.

cancer biology↗