SEARCH · Search bioRxiv

Results for “Cancer Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources

bioRxiv2017

Induction of DISE in ovarian cancer cells in vivo

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nano particles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

cancer biology↗

bioRxiv2017

CD95L mRNA And CD95L Derived si- and shRNAs Kill Cancer Cells Through An RNAi Mechanism By Targeting Survival Genes

Over 80% of multiple tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that using specific toxic RNAi-active sequences present in the genome can kill cancer cells.

cancer biology↗

bioRxiv2017

Sepsis Increases Perioperative Metastases In A Murine Model

BackgroundCancer surgery can promote tumour metastases and worsen prognosis, but the effects of perioperative complications such as sepsis, blood loss, and hypothermia on subsequent cancer metastases have not been addressed.\n\nObjectiveTo evaluate the effect of common perioperative factors on postoperative tumour metastases in murine models of cancer surgery. We hypothesize that perioperative blood loss, hypothermia, and sepsis facilitate tumour metastases in these murine models.\n\nMethodsPrior to surgery, pulmonary metastases were established by intravenous challenge of CT26LacZ colon cancer cells in Balb/c mice or B16LacZ melanoma cells in C57Bl/6 mice. Surgical stress was generated through partial hepatectomy (PH) or left nephrectomy (LN). Sepsis was induced by puncturing the cecum to express stool into the abdomen. Hemorrhagic shock was induced by removal of 30% of total blood volume via saphenous vein. Hypothermia was induced by removing the heating apparatus during surgery and lowering core body temperatures to 30{degrees}C. Lung tumour burden was quantified 3 days following surgery.\n\nResultsSurgically stressed mice subjected to Stage 3 hemorrhagic shock or hypothermia did not show an additional increase in lung tumour burden. In contrast, surgically stressed mice subjected to intraoperative sepsis demonstrated an additional 2-fold increase in the number of tumour metastases. Furthermore, natural killer (NK) cell function, as assessed by YAC-1 tumour cell lysis, was significantly attenuated in surgically stressed mice subjected to intraoperative sepsis. Both NK cell-mediated cytotoxic function and lung metastases were improved with perioperative administration of polyI:C, which is a ligand for toll-like receptor (TLR)-3\n\nConclusionsPerioperative sepsis, but not hemorrhagic shock or hypothermia, enhances the prometastatic effect of surgery in murine models of cancer. Identification of the mechanisms underlying perioperative immune suppression will be critical in the development of immunomodulation strategies that aim to attenuate perioperative metastatic disease.

cancer biology↗

bioRxiv2017

RANKL Signaling Sustains Primary Tumor Growth In Genetically Engineered Mouse Models Of Lung Adenocarcinoma

HypothesisNon-small cell lung cancer (NSCLC) is the leading cause of cancer mortality. Recent retrospective clinical analyses suggest that blocking the receptor activator of NF-{kappa}B (RANK) signaling pathway inhibits the growth of NSCLC and might represent a new treatment strategy.\n\nMethodsRANK and RANKL expression in human lung adenocarcinoma was interrogated from publicly available gene expression datasets. Several genetically engineered mouse models were used to evaluate treatment efficacy of RANK-Fc to block RANKL, with primary tumor growth measured longitudinally using micro-computed tomography. A combination of RANKL blockade with cisplatin was tested to mirror an ongoing clinical trial.\n\nResultsIn human lung adenocarcinoma datasets, RANKL expression was associated with decreased survival and KRAS mutation, with the highest levels in tumors with co-occurring KRAS and LKB1 mutations. In KrasLSL-G12D/WT, KrasLSL-G12D/WT; Lkb1Flox/Flox and KrasLSL-G12D/WT; p53Flox/Flox mouse models of lung adenocarcinoma, we monitored an impaired progression of tumors upon RANKL blockade. Despite elevated expression of RANKL and RANK in immune cells, treatment response was not associated with major changes in the tumor immune microenvironment. Combined RANK-Fc with cisplatin revealed increased efficacy compared to single agents.\n\nConclusionsRANKL blocking agents impair the growth of primary lung tumors in several mouse models of lung adenocarcinoma, and suggest that patients with KRAS mutant lung tumors will benefit from such treatments.

cancer biology↗

bioRxiv2017

Postmitotic Separation Enables Selective Niche Retention Of One Daughter Cell In Intestinal Crypts And Is Facilitated By Interkinetic Nuclear Migration And Basal Tethering

Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particullary important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to position. To determine how position is achieved we followed proliferating cells in intestinal organoids and discovered that behaviour of mitotic sisters predicted long-term positioning. Normally, 70% of sisters remain neighbours while 30% lose contact separating after cytokinesis. Postmitotic placements predict differences in positions of sisters later: adjacent sisters reach similar positions; one separating sister remains close to its birthplace, the other moves upward. Computationally modelling crypt dynamics confirmed post-mitotic separation as a mechanism for placement of sisters into different niches. Separation depends on interkinetic nuclear migration, cell size, and asymmetric tethering by a basal process. These processes are altered when Adenomatous polyposis coli (Apc) is mutant and separation is lost. We conclude that post-mitotic placement enables stochastic niche exit and when defective, supports the clonal expansion of Apc mutant cells.

cancer biology↗

bioRxiv2017

Repurposing Tofacitinib As An Anti-Myeloma Therapeutic To Reverse Growth-Promoting Effects Of The Bone Marrow Microenvironment

The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Interleukin-6 (IL-6) and downstream JAK/STAT signaling are thought to be central components of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor FDA-approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Here, we validated both in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated murine xenograft models of myeloma, that tofacitinib showed both single-agent and combination therapeutic efficacy in myeloma models. Surprisingly, we found that ruxolitinib, an FDA-approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. RNA-seq and unbiased phosphoproteomics revealed that marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma plasma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib specifically reverses the growth-promoting effects of the tumor microenvironment through blocking an IL-6-mediated signaling axis. As tofacitinib is already FDA-approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.

cancer biology↗

bioRxiv2017

Contextual Tumor Suppressor Function Of T Cell Death-Associated Gene 8 (TDAG8) In Hematological Malignancies

Key points1. TDAG8 (GPR65) gene expression is down-regulated in hematological malignancies.\n\n2. Restoration of TDAG8 gene expression in blood cancer cells suppresses tumor growth and metastasis.\n\nAbstractExtracellular acidosis is a condition found within the tumor microenvironment due to inadequate blood perfusion, hypoxia, and altered tumor cell metabolism. Acidosis has pleiotropic effects on malignant progression; therefore it is essential to understand how acidosis exerts its diverse effects. In this study a bioinformatics analysis revealed the expression of the proton sensing G-protein-coupled receptor TDAG8 is significantly reduced in human blood cancers in comparison to normal blood cells. To understand how TDAG8 functions in hematological malignancies, TDAG8 expression was restored in U937 acute myeloid leukemia cells and other blood cancer cells. It was discovered that severe acidosis, pH 6.4, inhibited U937 cell proliferation while mild acidosis, pH 6.9, stimulated cell proliferation. However, restoring TDAG8 gene expression modulated the U937 cell response to mild extracellular acidosis and physiological pH by reducing cell proliferation. Tumor xenograft experiments further revealed that restoring TDAG8 expression in U937 and Ramos cancer cells reduced tumor growth. It was also shown U937 cells with restored TDAG8 expression attached less to Matrigel, migrated slower toward a chemoattractant, and metastasized less in severe combined immunodeficient mice. These effects correlated with a reduction in c-myc oncogene expression. The mechanistic investigation indicated that G13/RhoA signaling arbitrated the TDAG8-mediated c-myc oncogene repression in response to acidosis. Overall, this study provides compelling data to support the concept that TDAG8 functions as a contextual tumor suppressor in hematological malignancies and sensitizes blood cancer cells to acidotic stress.

cancer biology↗

bioRxiv2017

Paracrine Activin-A Signaling Promotes Melanoma Growth And Metastasis Through Immune Evasion

The secreted growth factor Activin-A of the TGF{beta} family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor II B as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that while cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting anti-tumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

cancer biology↗

bioRxiv2017

Significant Associations Between Driver Gene Mutations And DNA Methylation Alterations Across Many Cancer Types

Recent evidence shows that mutations in several driver genes can cause aberrant methylation patterns, a hallmark of cancer. In light of these findings, we hypothesized that the landscapes of tumor genomes and epigenomes are tightly interconnected. We measured this relationship using principal component analyses and methylation-mutation associations applied at the nucleotide level and with respect to genome-wide trends. We found a few mutated driver genes were associated with genome-wide patterns of aberrant hypomethylation or CpG island hypermethylation in specific cancer types. We identified associations between 737 mutated driver genes and site-specific methylation changes. Moreover, using these mutation-methylation associations, we were able to distinguish between two uterine and two thyroid cancer subtypes. The driver gene mutation-associated methylation differences between the thyroid cancer subtypes were linked to differential gene expression in JAK-STAT signaling, NADPH oxidation, and other cancer-related pathways. These results establish that driver-gene mutations are associated with methylation alterations capable of shaping regulatory network functions. In addition, the methodology presented here can be used to subdivide tumors into more homogeneous subsets corresponding to their underlying molecular characteristics, which could improve treatment efficacy.\n\nAuthor summaryMutations that alter the function of driver genes by changing DNA nucleotides have been recognized as a key player in cancer progression. Recent evidence showed that DNA methylation, a molecular signature that is used for controlling gene expression and that consists of cytosine residues with attached methyl groups in the context of CG dinucleotides, is also highly dysregulated in cancer and contributes to carcinogenesis. However, whether those methylation alterations correspond to mutated driver genes in cancer remains unclear. In this study, we analyzed 4,302 tumors from 18 cancer types and demonstrated that driver gene mutations are inherently connected with the aberrant DNA methylation landscape in cancer. We showed that those driver gene-associated methylation patterns can classify heterogeneous tumors in a cancer type into homogeneous subtypes and have the potential to influence the genes that contribute to tumor growth. This finding could help us to better understand the fundamental connection between driver gene mutations and DNA methylation alterations in cancer and to further improve the cancer treatment.

cancer biology↗

bioRxiv2017

Multiple antibodies identify Glypican-1 on serum exosomes from patients with pancreatic cancer

Exosomes are man-sized vesicles shed by all cells, including cancer cells. Exosomes can serve as novel liquid biopsies for diagnosis of cancer with potential prognostic value. The exact mechanism/s associated with sorting or enrichment of cellular components into exosomes are still largely unknown. We reported Glypican-1 (GPC1) on the surface of cancer exosomes and provided evidence for the enrichment of GPC1 in exosomes from patients with pancreatic cancer1. Several different laboratories have validated this novel conceptual advance and reproduced the original experiments using multiple antibodies from different sources. These include anti-GPC1 antibodies from ThermoFisher (PA5-28055 and PA-5-24972)1,2, Sigma (SAB270028), Abnova (MAB8351, monoclonal antibodies clone E9E)3, EMD Millipore (MAB2600-monoclonal antibodies)4, SantaCruz5, and R&D Systems (BAF4519)2. This report complements such independent findings and report on the specific detection of Glypican-1 on the exosomes derived from the serum of pancreas cancer patients using multiple antibodies. Additionally, the specificity of the antibodies to GPC1 was determined by western blot and Protein Simple analyses of pancreatic cancer cells and their exosomes. Interestingly, our results highlight a specific enrichment of high molecular weight GPC1 on exosomes, potentially contributed by heparan sulfate and other glycosylation modifications.

cancer biology↗

bioRxiv2017

Global Cancer Transcriptome Quantifies Repeat Element Polarization Between Immunotherapy Responsive and T cell Suppressive Classes

Growing evidence indicates that innate immune pathway activation is critical for responses to immunotherapy and overall cancer prognosis. It has been posited that innate immunity in the tumor microenvironment can be driven by derepression of endogenous repetitive element RNA. The ability to characterize these species can potentially provide novel predictive biomarkers for tumor immune responses and a mechanistic basis for elements of innate activation by tumors. We first compared total RNA and poly(A)-capture protocols applied to tumor RNA-sequencing to detect non-RNA coding transcriptomes in the TCGA. While the poly(A) protocol efficiently detects coding, most non-coding genes, and much of the LINE/SINE/ERV repeat repertoire, we found that it fails to capture overall repeat expression and co-expression. The probing of total RNA expression reveals distinct repetitive co-expression subgroups. Secondly, we found that total repeat element expression delivers the most dynamic changes in samples, which may serve as more robust biomarkers of clinical outcomes. Finally, we show that while expression of ERVs, but not other immunostimulatory repeats such as HSATII, is associated with response to immunotherapy in a cohort of patients with urothelial cancer treated with anti-PD-L1 therapy, global repeat derepression strongly correlates with an immunosuppressive phenotype in the microenvironment of colorectal and pancreatic tumors. We validate in situ in human primary tumors, associating the immunosuppressive phenotype with HSATII expression. In conclusion, we demonstrate the importance of analyzing repetitive element RNAs as potential biomarkers of response to immunotherapy and the need to better characterize these features in next generation sequencing protocols.

cancer biology↗

bioRxiv2017

Non-classical estrogen signaling inhibits melanoma and improves response to PD-1 blockade

Female sex and pregnancy are associated with reduced risk of melanoma and improved stage specific survival; however, the mechanism underlying this apparent clinical benefit is unknown. We previously discovered that pregnancy-associated 17{beta}-estradiol drives melanocyte differentiation by activating the nonclassical G-protein coupled estrogen receptor (GPER). Here, we show that pregnancy inhibits melanoma, and that transient GPER activation induces long-term changes in melanocytes, which are associated with increased cellular differentiation and resistance to melanoma. A selective GPER agonist induced c-Myc protein degradation, slowed tumor growth, and inhibited expression of immune suppressive proteins including PD-L1, suggesting that GPER signaling may render melanoma cells more vulnerable to immunotherapy. Systemically delivered GPER agonist was well tolerated, and cooperated synergistically with PD-1 blockade in melanoma-bearing mice to dramatically extend survival. These results thus define GPER as a target for differentiation-based melanoma therapy.\n\nSignificanceImmune checkpoint inhibitors including PD-1 induce durable remissions in only 30% of patients with advanced melanoma. This work demonstrates that PD-1 efficacy is significantly improved by systemic delivery of a selective agonist of the nonclassical estrogen receptor, GPER, which drives melanoma differentiation and renders tumors more vulnerable to the immune system.

cancer biology↗

bioRxiv2017

An anti-cancer binary system activated by bacteriophage HK022 Integrase

Cancer gene therapy is a great promising tool for cancer therapeutics due to the specific targeting based on the cancerous gene expression background. Binary systems based on site-specific recombination are one of the most effective potential approaches for cancer gene therapy. In these systems, a cancer specific promoter expresses a site-specific recombinase/integrase that in turn controls the expression of a toxin gene. In the current study, we have developed a new HK022 bacteriophage Integrase (Int) based binary system activating a Diphtheria toxin (DTA) gene expression specifically in cancer cells. We have demonstrated the efficiency, and the high specificity of the system in vitro in cell cultures and in vivo in a lung cancer mouse model. Strikingly, different apoptotic and anti-apoptotic factors demonstrated a remarkable efficacy killing capability of the Int-based binary system compared to the conventional hTERT-DTA mono system in the LLC-Kat lung cancer mice model; we observed that the active hTERT promoter down regulation by the transcription factors Mad-1 is the cornerstone of this phenomenon. The new Int-based binary system offers advantages over already known counterparts and may therefore be developed into a safer and efficient cancer treatment technology.

cancer biology↗

bioRxiv2017

Multi-dimensional genomic analysis of myoepithelial carcinoma identifies prevalent oncogenic gene fusions

Myoepithelial carcinoma (MECA) is an aggressive type of salivary gland cancer with largely unknown molecular features. MECA may arise de novo or result from oncogenic transformation of a pre-existing pleomorphic adenoma (MECA ex-PA). We comprehensively analyzed the molecular alterations in MECA with integrated genomic analyses. We identified a low mutational load (0.5/MB), but a high prevalence of fusion oncogenes (28/40 tumors; 70%). We found FGFR1-PLAG1 in 7 (18%) cases, and the novel TGFBR3-PLAG1 fusion in 6 (15%) cases. TGFBR3-PLAG1 was specific for MECA de novo tumors or the malignant component of MECA ex-PA, was absent in 723 other salivary gland tumors, and promoted a tumorigenic phenotype in vitro. We discovered other novel PLAG1 fusions, including ND4-PLAG1, which is an oncogenic fusion between mitochondrial and nuclear DNA. One tumor harbored an MSN-ALK fusion, which was tumorigenic in vitro, and targetable with ALK inhibitors. Certain gene fusions were predicted to result in neoantigens with high MHC binding affinity. A high number of copy number alterations was associated with poorer prognosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future steps of diagnostic and therapeutic research in this lethal cancer.

cancer biology↗

bioRxiv2017

The effects of mutational process and selection on driver mutations across cancer types

Epidemiological evidence has long associated environmental mutagens with increased cancer risk. However, links between specific mutation-causing processes and the acquisition of individual driver mutations have remained obscure. Here we have used public cancer sequencing data to infer the independent effects of mutation and selection on driver mutation complement. First, we detect associations between a range of mutational processes, including those linked to smoking, ageing, APOBEC and DNA mismatch repair (MMR) and the presence of key driver mutations across cancer types. Second, we quantify differential selection between well-known alternative driver mutations, including differences in selection between distinct mutant residues in the same gene. These results show that while mutational processes play a large role in determining which driver mutations are present in a cancer, the role of selection frequently dominates.

cancer biology↗

bioRxiv2017

The likelihood of heterogeneity or additional mutation in KRAS or associated oncogenes to compromise targeting of oncogenic KRAS G12C

Activating mutations in RAS genes are associated with approximately 20% of all human cancers. New targeted therapies show preclinical promise in inhibiting the KRAS G12C variant, however, concerns exist regarding the effectiveness of such therapies in vivo given the possibilities of existing intratumor heterogeneity or de novo mutation leading to treatment resistance. We performed deep sequencing of 27 KRAS G12 positive lung tumors and found no evidence of other oncogenic mutations within KRAS or within commonly mutated downstream genes that could confer resistance at the time of treatment. Furthermore, we estimate the de novo mutation rate in KRAS position 12 and in genes downstream of KRAS. We find that mutations that confer resistance are about as likely to occur downstream of KRAS as within KRAS. Moreover, we present an approach for estimation of the selection intensity for these point mutations that explains their high prevalence in tumors. Our approach predicts that BRAF V600E would provide the highest fitness advantage for de novo resistant subclones. Overall, our findings suggest that resistance to targeted therapy of KRAS G12C positive tumors is unlikely to be present at the time of treatment and, among the de novo mutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage.\n\nOne Sentence SummaryMutations conferring resistance to KRAS G12C targeted therapy are unlikely to be present at the time of resection, and the likely mechanisms of evolved resistance are predicted be ones that are responsive to therapies that are in development or that are already available.

cancer biology↗

bioRxiv2017

The polyploid state plays a tumor suppressive role in the liver

Most cells in the liver are polyploid, but the functional role of polyploidy is unknown. Polyploidization normally occurs through cytokinesis failure and endoreduplication around the time of weaning. To interrogate the function of polyploidy while avoiding irreversible manipulations of essential cell cycle genes, we developed multiple orthogonal mouse models to transiently and potently alter liver ploidy. Premature weaning, as well as in vivo knockdown of E2f8 or Anln, allowed us to toggle between diploid and polyploid states. While there was no impact of ploidy alterations on liver function, metabolism, or regeneration, hyperpolyploid mice suppressed and hyperdiploid mice accelerated tumorigenesis in mutagen and high fat induced models. Mechanistically, the diploid state was more susceptible to Cas9-mediated tumor suppressor loss but was similarly susceptible to MYC oncogene activation, indicating that ploidy differentially protected the liver from distinct genomic aberrations. Our work suggests that polyploidy evolved to prevent malignant outcomes of liver injury.

cancer biology↗

bioRxiv2017

Impairment of mitochondrial respiratory function as an early biomarker of apoptosis induced by growth factor removal

Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40% inhibition. A slight increase in respiration following addition of cytochrome c, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.

cancer biology↗