Transformed epithelia trigger non-tissue-autonomous tumor suppressor response by adipocytes via activation of toll and Eiger/TNF signaling

High tumor burden is associated with increased levels of circulating inflammatory cytokines that influence the pathophysiology of the tumor and its environment. The cellular and molecular events mediating the organismal response to a growing tumor are poorly understood. Here, we report a bidirectional crosstalk between epithelial tumors and the fat body—a peripheral immune tissue—in Drosophila. Tumors trigger a systemic immune response through activation of Eiger/TNF signaling, which leads to Toll pathway upregulation in adipocytes. Reciprocally, Toll elicits a non-tissue-autonomous program in adipocytes, which drives tumor cell death. Hemocytes play a critical role in this system by producing the ligands Spätzle and Eiger, which are required for Toll activation in the fat body and tumor cell death. Altogether, our results provide a paradigm for a long-range tumor suppression function of adipocytes in Drosophila, which may represent an evolutionarily conserved mechanism in the organismal response to solid tumors

The in vivo function of the p53 target gene TIGAR

The p53 tumour suppressor inhibits tumour development via various mechanisms such as apoptosis, inhibition of proliferation or the activation of senescence. Recently, several studies have indicated a novel role of p53 in the regulation of energy metabolism. Previously we have discovered TIGAR, a p53 target gene that acts as a fructose-2,6-bisphosphatase. TIGAR therefore can redirect glucose from the glycolytic pathway to the pentose phosphate pathway (PPP), which promotes NADPH production to generate reduced glutathione for protecting against ROS, and also ribose 5 phosphate production for nucleotide synthesis. In order to understand the function of TIGAR in vivo, we generated TIGAR deficient mice. We have determined a critical role of TIGAR in rapidly proliferating tissue, either for repair after damage or during tumor development

Development of an inducible mouse model of iRFP713 to track recombinase activity and tumour development in vivo

While the use of bioluminescent proteins for molecular imaging is a powerful technology to further our understanding of complex processes, fluorescent labeling with visible light fluorescent proteins such as GFP and RFP suffers from poor tissue penetration and high background autofluorescence. To overcome these limitations, we generated an inducible knock-in mouse model of iRFP713. This model was used to assess Cre activity in a Rosa Cre-ER background and quantify Cre activity upon different tamoxifen treatments in several organs. We also show that iRFP can be readily detected in 3D organoid cultures, FACS analysis and in vivo tumour models. Taken together we demonstrate that iRFP713 is a progressive step in in vivo imaging and analysis that widens the optical imaging window to the near-infrared spectrum, thereby allowing deeper tissue penetration, quicker image acquisition without the need to inject substrates and a better signal to background ratio in genetically engineered mouse models (GEMMs)

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, levels of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts have been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis

Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities

The initiator methionine tRNA drives secretion of type II collagen from stromal fibroblasts to promote tumor growth and angiogenesis

Summary: Expression of the initiator methionine tRNA (tRNAi Met) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi Met expression levels influence tumor progression. We have found that tRNAi Met expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi Met in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi Met contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi Met gene (2+tRNAi Met mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi Met mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi Met mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi Met significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi Metoverexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl- 3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi Met-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi Met mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi Met levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult <i>Drosophila</i> midgut

Background: Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream.&lt;p&gt;&lt;/p&gt; Results: Here we report a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the &lt;i&gt;Drosophila&lt;/i&gt; midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP.&lt;p&gt;&lt;/p&gt; Conclusions: We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.&lt;p&gt;&lt;/p&gt

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double strand breaks (DSB) which co-localized with 'replication factories' and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (&gt;1Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell-specific target with significant clinical potential

Pharmacokinetics, safety and tolerability of olaparib and temozolomide for recurrent glioblastoma: results of the phase I OPARATIC trial

Background: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide chemotherapy in pre-clinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side-effects of temozolomide. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib, and assess the safety and tolerability of its combination with temozolomide. Methods: Pre-clinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose temozolomide in a 3+3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumour core and tumour margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results: Olaparib was a substrate for multi-drug resistance protein-1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients, median 496nM) and 21/21 tumor margin specimens (9 patients, median 512.3nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150mg (3 days/week) with TMZ 75mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression-free at 6 months. Olaparib radiosensitized six glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusions: Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better pre-clinical models

Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation

Objective: Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development.&lt;p&gt;&lt;/p&gt; Design: LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development.&lt;p&gt;&lt;/p&gt; Results: LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitis-associated colorectal mouse cancer model.&lt;p&gt;&lt;/p&gt; Conclusions: This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells.&lt;p&gt;&lt;/p&gt