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Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming.One-Sentence SummaryAfter severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

Tumor growth and metastasis involve a complex relationship with the tissue microenvironment. A proliferating tumor encounters several stresses from the microenvironment like hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells can co-opt existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR involves transcriptional and translational activation of signaling pathways designed to relieve cellular stress and attenuate cell death; although, in the case of unresolved or chronic ER stress UPR can promote cell death. However, the mechanisms by which the UPR influences cell fate in the presence of ER stress are poorly understood. To address this, we have used a functional CRISPR-based genetic knockout screen, to determine novel regulators of UPR and the mechanisms involved to control cellular fate following chronic ER stress. We delivered a lentiviral genome-wide CRISPR-Cas9 knockout library to two cell lines and identified sgRNAs which are over-represented (pro-apoptotic genes), or underrepresented (pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the highest-ranking gene candidates was Survivin/BIRC5, a protein which is overexpressed in tumor cells compared to normal tissues. Survivin was first identified as an inhibitor of apoptosis but also participates in the regulation of the cell cycle. Survivin held a high position in the negative ranking scale of all screens suggesting a possible prosurvival role in response to ER stress. In agreement, our results show that genetic knock down or chemical inhibition of Survivin led to sensitization of cells to ER stress. Moreover, both in vitro an in vivo data support that PERK (a key sensor of the third arm of the UPR cascade) is responsible for this sensitization. Interestingly, we observed that ER stress in turn leads to downregulation of Survivin in a dose and time dependent fashion, both in the level of protein but also RNA, employing a variety of mechanisms like protein translation, degradation and RNA synthesis. Foreseeably, PERK knock down reverses the aforementioned effect supporting its role as a key participant in the interplay between Survivin and ER stress. Though the balance between those two is still to be understood our results demonstrate that Survivin holds a novel function as an ER stress regulator but also targeting of PERK and Survivin in a clinical setting could reveal new windows in therapeutic intervention in malignancy. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Jerome Lin, Giorgos Skoufos, Ioannis Verginadis, Artemis Chatzigeorgiou, Constantinos Koumenis. Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 144.

ATF4 is a major effector of the Integrated Stress Response (ISR), a homeostatic mechanism coupling cell growth and survival to bioenergetic demands. Although the pro-tumorigenic role of the ISR in a tumor cell-intrinsic manner has been established, its role in cell-extrinsic processes remains unexplored. Using novel conditional knockout ATF4 mouse models, we show that global, or fibroblast (FB)-specific loss of host ATF4 results in abnormal tumor vascularization and a pronounced tumor growth delay in syngeneic melanoma and pancreatic tumor models, a phenotype which is largely reversed by co-injection of ATF4wt/wt FBs. Single-cell tumor transcriptomics uncovered a reduction of markers associated with FB activation in a cluster of perivascular cancer-associated fibroblasts (CAF) in ATF4Δ/Δ mice. ATF4Δ/Δ FBs displayed significant defects in collagen biosynthesis and deposition and reduced ability to support angiogenesis in vitro. Mechanistically, ATF4 directly regulates the expression of the Col1a1 gene as well as the biosynthesis of glycine and proline, the major amino acids comprising collagen fibers. Analysis of human tumor samples revealed a strong correlation between ATF4 and collagen levels and between an ATF4 FB signature and expression of collagen genes. Our findings uncover a novel role of stromal ATF4 in shaping CAF functionality, a key driver of disease progression and therapy resistance.

Since the initial success of cisplatin, metal complexes and organometallic compounds have been gaining growing interest in cancer therapy. It is well known that organotin(IV) compounds display strong biological activity. The triorganotin compound [(C(6)H(5))(3)Sn(cmbzt)] (cmbzt=5-chloro-2-mercaptobenzothiazole) (SnCMB), was tested for its antiproliferative and antitumour activities. Two sets of experimental procedures were followed: (1) In vitro and ex vivo procedures included the study of the cytotoxic activity of the complex against leiomyosarcoma cells (LMS) and on a normal human fibroblast line (MRC5) by the MTT assay (cell proliferation), colony formation efficiency and flow cytometric analysis with Annexin V-FITC. The anticoagulation properties of the complex were also studied. (2) In vivo procedures included acute toxicity studies and finally administration of the complex to tumour bearing Wistar rats. The results showed that the complex exhibited potent cytotoxic activity (LMS IC(50)=155 nM) and induced significant apoptosis against LMS cells. Acute toxicity studies on Wistar rats presented kidney and liver toxicity at a single dose of 40 mg/kg body wt. Furthermore, antitumour activity studies on sarcoma bearing Wistar rats revealed that SnCMB complex, administrated in two different therapeutic schemes (treated with 4 × 2 mg/kg body wt every 5 days and 3 × 2.67 mg/kg body wt every 10 days of SnCMB complex), prolonged mean survival time (by 50% and 70% respectively), but failed to decrease the mean tumour growth rate (MTGR) compared to the control group (p0.01). In conclusion, the organic complex SnCMB possess potent cytotoxic and antimetastatic effects, and low toxicity introducing it as possible successor of organometallic compounds used nowadays in chemotherapy.

In the present study, the effects of a resonant low intensity static electromagnetic field (EMF), causing no thermal effects, on Wistar rats have been investigated. Sarcoma cell lines were isolated from leiomyosarcoma tumors induced in Wistar rats by the subcutaneous (s.c) injection of 3,4-benzopyrene. Furthermore, smooth muscle cells (SMC) were isolated from the aorta of Wistar rats and cultivated. Either leiomyosarcoma cells (LSC) or SMC were used to record a number of characteristic resonant radiofrequencies, in order to determine the specific electromagnetic fingerprint spectrum for each cell line. These spectra were used to compose an appropriate algorithm, which transforms the recorded radiofrequencies to emitted ones. The isolated LSC were cultured and then exposed to a resonant low intensity radiofrequency EMF (RF-EMF), at frequencies between 10 kHz to 120 kHz of the radiowave spectrum. The exposure lasted 45 consecutive minutes daily, for two consecutive days. Three months old female Wistar rats were inoculated with exposed and non-exposed to EMF LSC (4 x 10(6) LCS for animal). Inoculated with non-exposed to EMF cells animals were then randomly separated into three Groups. The first Group was sham exposed to the resonant EMF (control Group-CG), the second Group after the inoculation of LSC and appearance of a palpable tumor mass, was exposed to a non-resonant EMF radiation pattern, for 5 h per day till death of all animals (experimental control Group-ECG). The third Group of animals after inoculation of LSC and the appearance of a palpable tumor mass, was exposed to the resonant EMF radiation for 5 h per day, for a maximum of 60 days (experimental Group-I, EG-I). A fourth Group of animals was inoculated with LSC exposed to EMF irradiation and were not further exposed to irradiation (experimental Group-II, EG-II). Tumor induction was 100% in all Groups studied and all tumors were histologically identified as leiomyosarcomas. In the case of the EG-I, a number of tumors were completely regretted (final tumor induction: 66%). Both Groups of animals inoculated with exposed or non-exposed to the EMF LSC, (EG-I and EG-II, respectively) demonstrated a significant prolongation of the survival time and a lower tumor growth rate, in comparison to the control Group (CG) and the experimental control Group (ECG). However, the survival time of EG-I animals was found to be significantly longer and tumor growth rate significantly lower compared to EG-II animals. In conclusion, our results indicate a specific anticancer effect of resonant EMF irradiation. These results may possibly be attributed to (a) the duration of exposure of LSC and (b) the exposure of the entire animal to this irradiation.

In this study we investigated the effects of low intensity static radiofrequency electromagnetic field (EMF) causing no thermal effects, on leiomyosarcoma cells (LSC), isolated from tumors of fifteen Wistar rats induced via a 3,4-benzopyrene injection. Electromagnetic resonance frequencies measurements and exposure of cells to static EMF were performed by a device called multi channel dynamic exciter 100 V1 (MCDE). The LSC were exposed to electromagnetic resonance radiofrequencies (ERF) between 10 kHz to 120 kHz, for 45 min. During a 24h period, after the exposure of the LSC to ERF, there was no inhibition of cells proliferation. In contrast, at the end of a 48 h incubation period, LSC proliferation dramatically decreased by more than 98% (P0.001). At that time, the survived LSC were only 2% of the total cell population exposed to ERF, and under the same culture conditions showed significant decrease of proliferation. These cells were exposed once again to ERF for 45 min (totally 4 sessions of exposure, of 45 min duration each) and tested using a flow cytometer. Experiments as above were repeated five times. It was found that 45% of these double exposed to ERF, LSC (EMF cells) were apoptotic and only a small percentage 2%, underwent mitosis. In order to determinate their metastatic potential, these EMF cells were also counted and tested by an aggregometer for their ability to aggregate platelets and found to maintain this ability., since they showed no difference in platelet aggregation ability compared to the LSC not exposed to ERF (control cells). In conclusion, exposure of LSC to specific ERF, decreases their proliferation rate and induces cell apoptosis. Also, the LSC that survived after exposed to ERF, had a lower proliferation rate compared to the LSC controls (P0.05) but did not loose their potential for metastases (platelet aggregation ability). The non-malignant SMC were not affected by the EMF exposure (P0.4). The specific ERF generated from the MCDE electronic device, used in this study, is safe for humans and animals, according to the international safety standards.