Basic Caenorhabditis Elegans Methods: Synchronization And Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism (1). C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years (2). Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved (3). C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage (4,5) together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology (6), aging (7,8), stem cell biology (9) and germ line biology (10). An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4', 6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them

The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

Heterotypic interactions between newly transformed cells and normal surrounding cells define tumor's fate in incipient carcinomas. Once homeostasis has been lost, normal resident fibroblasts become carcinoma-associated fibroblasts, conferring protumorogenic properties on these normal cells. Here we describe the IL1 beta-mediated interplay between cancer cells and normal colonic myofibroblasts (NCFs), which bestows differential sensitivity to cytotoxic drugs on tumor cells. We used NCFs, their conditioned media (CM), and cocultures with tumor cells to characterize the IL1 beta-mediated crosstalk between both cell types. We silenced IL1 beta in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1 beta is overexpressed in cocultured tumor cells. IL1 beta enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1 beta-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGF beta 1-driven NCFs. IL1 beta induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1 beta-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1 beta-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells. Keyword

Requirement for epithelial p38α in KRAS-driven lung tumor progression

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals

Circular RNA CpG island hypermethylation-associated silencing in human cancer

Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs (lncRNAs), participate in cellular transformation. Work done in the last decade has also demonstrated that ncRNAs with growth-inhibitory functions can undergo promoter CpG island hypermethylation-associated silencing in tumorigenesis. Herein, we wondered whether circular RNAs (circRNAs), a type of RNA transcripts lacking 5′-3′ ends and forming closed loops that are gaining relevance in cancer biology, are also a target of epigenetic inactivation in tumors. To tackle this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in conjuction with circRNA expression microarrays. We have found that the loss of DNA methylation provokes a release of circRNA silencing. In particular, we have identified that promoter CpG island hypermethylation of the genes TUSC3 (tumor suppressor candidate 3), POMT1 (protein O-mannosyltransferase 1), ATRNL1 (attractin-like 1) and SAMD4A (sterile alpha motif domain containing 4A) is linked to the transcriptional downregulation of both linear mRNA and the hosted circRNA. Although some circRNAs regulate the linear transcript, we did not observe changes in TUSC3 mRNA levels upon TUSC3 circ104557 overexpression. Interestingly, we found circRNA-mediated regulation of target miRNAs and an in vivo growth inhibitory effect upon TUSC3 circ104557 transduction. Data mining for 5′-end CpG island methylation of TUSC3, ATRNL1, POMT1 and SAMD4A in cancer cell lines and primary tumors showed that the epigenetic defect was commonly observed among different tumor types in association with the diminished expression of the corresponding transcript. Our findings support a role for circRNA DNA methylation-associated loss in human cancer

Sensitization of retinoids and corticoids to epigenetic drugs in MYC-activated lung cancers by antitumor reprogramming

Components of the SWI/SNF chromatin remodeling complex, including BRG1 (also SMARCA4), are inactivated in cancer. Among other functions, SWI/SNF orchestrates the response to retinoid acid (RA) and glucocorticoids (GC) involving downregulation of MYC. The epigenetic drugs SAHA and azacytidine, as well as RA and GC, are currently being used to treat some malignancies but their therapeutic potential in lung cancer is not well established. Here we aimed to determine the possible therapeutic effects of azacytidine and SAHA (A/S) alone or in combination with GC plus RA (GC/RA) in lung cancers with either BRG1 inactivation or MYC amplification. In vitro, responses to GC/RA treatment were more effective in MYC-amplified cells. These effects were mediated by BRG1 and involved a reprogramming towards prodifferentiation gene expression signatures and downregulation of MYC. In MYC-amplified cells, administration of GC/RA enhanced the cell growth inhibitory effects of A/S which, in turn, accentuated the prodifferentiation features promoted by GC/RA. Finally, these treatments improved overall survival of mice orthotopically implanted with MYC-amplified, but not BRG1-mutant, cells and reduced tumor cell viability and proliferation. We propose that the combination of epigenetic treatments with retinoids and corticoids of MYC-driven lung tumors constitute a strategy for therapeutic intervention in this otherwise incurable disease

Hypersaline Infusion Protocol through the Portal Vein may Focus Electroporation on Tumor Tissue, but is it really Safe? Preliminary Results

Introduction: Irreversible Electroporation (IRE) is highly dependent on the electrical conductivity of the tissue and the high conductivity of tumor tissue, which leads to a lower field than in the surrounding healthy tissue. Hypersaline Infusion (HI) through the portal vein focuses IRE on scattered liver tumors, by creating a differential conductivity between the different types of tissue. The aim of this study is to determine the effects of the HI protocol on the hepatic and histological biochemical results. Methods: Ten male Sprague Dawley rats were used for HI protocol. Blood samples were collected at pre-, immediately post-, 24-hrs, 72-hrs, 1-week and 3-weeks post-HI. All the animals were sacrificed after one-month follow-up in order to collect histological samples. Results: The mortality rate in this procedure reached 30% (3/10). Only the pH and transaminases at 24-hrs were significantly and directly linked to mortality (p=0.036 and p=0.004, respectively). The three non-surviving animals had a four-time higher AST level at 24-hrs. Natremia normalized at 24-hrs post-HI. Statistically significant differences were found in hepatic necrosis between the non-surviving (n=3) and surviving rats (n=7) (30.67 ± 10.97 vs. 2.86 ± 7.56% respectively, p=0.01). Discussion: HI through the portal system involves a significant risk of possibly lethal cytolysis and acidosis. Therefore, compensatory measures and a reduced saline overload are warranted to improve the survival rates

The TGFβ pathway stimulates ovarian cancer cell proliferation by increasing IGF1R levels

In a search for new therapeutic targets for treating epithelial ovarian cancer, we analyzed the Transforming Growth Factor Beta (TGFβ) signaling pathway in these tumors. Using a TMA with patient samples we found high Smad2 phosphorylation in ovarian cancer tumoral cells, independently of tumor subtype (high-grade serous or endometrioid). To evaluate the impact of TGFβ receptor inhibition on tumoral growth, we used different models of human ovarian cancer orthotopically grown in nude mice (OVAs). Treatment with a TGFβRI&II dual inhibitor, LY2109761, caused a significant reduction in tumor size in all these models, affecting cell proliferation rate. We identified Insulin Growth Factor (IGF)1 receptor as the signal positively regulated by TGFβ implicated in ovarian tumor cell proliferation. Inhibition of IGF1R activity by treatment with a blocker antibody (IMC-A12) or with a tyrosine kinase inhibitor (linsitinib) inhibited ovarian tumoral growth in vivo. When IGF1R levels were decreased by shRNA treatment, LY2109761 lost its capacity to block tumoral ovarian cell proliferation. At the molecular level TGFβ induced mRNA IGF1R levels. Overall, our results suggest an important role for the TGFβ signaling pathway in ovarian tumor cell growth through the control of IGF1R signaling pathway. Moreover, it identifies anti-TGFβ inhibitors as being of potential use in new therapies for ovarian cancer patients as an alternative to IGF1R inhibition

RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery

Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development

Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence. Refractory/relapsed RMS patients present a bad prognosis that combined with the lack of specific biomarkers impairs the development of new therapies. Here, we utilize dynamic BH3 profiling (DBP), a functional predictive biomarker that measures net changes in mitochondrial apoptotic signaling, to identify anti-apoptotic adaptations upon treatment. We employ this information to guide the use of BH3 mimetics to specifically inhibit BCL-2 pro-survival proteins, defeat resistance and avoid relapse. Indeed, we found that BH3 mimetics that selectively target anti-apoptotic BCL-xL and MCL-1, synergistically enhance the effect of clinically used chemotherapeutic agents vincristine and doxorubicin in RMS cells. We validated this strategy in vivo using a RMS patient-derived xenograft model and observed a reduction in tumor growth with a tendency to stabilization with the sequential combination of vincristine and the MCL-1 inhibitor S63845. We identified the molecular mechanism by which RMS cells acquire resistance to vincristine: an enhanced binding of BID and BAK to MCL-1 after drug exposure, which is suppressed by subsequently adding S63845. Our findings validate the use of DBP as a functional assay to predict treatment effectiveness in RMS and provide a rationale for combining BH3 mimetics with chemotherapeutic agents to avoid tumor resistance, improve treatment efficiency, and decrease undesired secondary effects

Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the proapoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1. This article has an associated First Person interview with the first author of the paper