PO-300 Unveiling and exploiting cancer stem cell editing and immunogenicity for precision medicine

Introduction Immunogenic chemotherapy (IC) induces immunogenic cell death (ICD), which, similar to viral infection, leads to a cancer-cell autonomous Type-I-Interferon (IFN-I) signalling. This immunological signature is crucial for effective antitumor responses but may paradoxically promote the emergence of a rare population of cancer stem cells (CSCs) acting as a chemoresistant niche within the tumour and roots for metastasis and relapse. In this study, we have investigated the role of IFN-I during IC in inducing a cancer editing program resulting in the appearance of poor immunogenic CSCs. Material and methods Human and murine tumour cell lines were treated in vitro with ICD-inducers or IFN-I as control and the induction of CSC were analysed by cytofluorometry, quantitative real time (qRT)-PCR, 3D culture and functional assays. Free and vesicle-mediated nucleic acid transfer during ICD has been characterised by co-culture experiments. IC-induced CSC immunogenicity has been studied through cytofluorometry, microfluidic devices and in vivo experiments. All experiments have been done in triplicate and statistical significance evaluated by two-tailed Student's t test and two-way ANOVA. Results and discussions The transient/acute induction of IFN-I during ICD is followed by the appearance of a rare population of CSCs. Both free nucleic acids and extracellular vesicles are released during tumour ICD constituting the upstream inducers of IFN-I-mediated reprogramming of neighbouring cells. IC-induced CSCs display epithelial-to-mesenchymal transition traits, multidrug resistance and regenerative properties, and a significant tumorigenic potential when inoculated in immunodeficient and immunocompetent mice. As expected, tumour growth and size are reduced in the presence of an intact immune system. Experiments on microfluidic devices reveal a poor immunogenic potential of CSCs, further confirmed by the expression of immune checkpoint blockers. Conclusion Our results pinpoint a surprising link between ICD, IFN-I and CSCs. Elucidating the mechanisms of CSC editing together with a deep characterisation of CSC (immune) properties could be crucial to prevent tumour relapse. This could undoubtedly have dramatic implications for the clinical management of cancer in an era of terrific development of precision combined chemo-immune therapy

Replication stress in colorectal cancer stem cells

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po 288 replication stress response as a target for eradicating colorectal cancer stem cells

Introduction Cancer stem cells (CSCs) are subsets of multipotent SCs responsible for tumour development, propagation and evolution, whose targeting is required for tumour eradication. There is (pre)clinical evidence on a role of CSCs in therapeutic resistance and intra-tumour heterogeneity, which limits the efficacy of antineoplastic regimens. In this context, CSCs reportedly share with embryonic/adult SCs a very robust DNA damage response, which favours the survival and resistance to genotoxins, and can be exploited for therapeutic purposes. Material and methods We generated a panel of ~30 CRC patient-derived tumorspheres enriched for CSCs (CRC-SCs) and characterised them at the genetic level. To discover potential monotherapeutic anti-CSC agents, we performed high-throughput screenings on multiple CRC-SCs with a library of clinically-relevant drugs. Flow cytometry, fluorescence microscopy and epistatic analyses were conducted to uncover the mechanism of action of identified compound(s), while genetic, cytogenetic and phosphoproteomic studies were carried out to identify predictive biomarkers. DNA replication stress (RS) levels were evaluated by analysing phosphorylated ATM/RPA foci and by performing COMET and DNA fibre assays, and were modulated by single administration of genome destabilising agents or by prolonged exposure to increased doses of compounds targeting the replication stress response (RSR). Results and discussions We demonstrated that the CHK1 inhibitor LY2606368 is a potent anti-CSC agent able to kill more than one third of CRC-SCs, both in vitro and in vivo. Moreover, we provided evidence of high but heterogeneous RS levels in CRC-SCs, showing that, in CRC-SCs, RS is mainly boosted endogenously by p53 deficiency, supernumerary chromosomes and DNA replication abnormalities, which results in high dependency on CHK1-mediated RSR. Accordingly, formerly LY2606368-resistant CRC-SCs were sensitised by boosting DNA replication errors or inducing whole-genome doubling, while formerly LY2606368-sensitive CRC-SCs made resistant by the continuous in vitro or in vivo administration of LY2606368 displayed diminished RS levels due to RSR rewiring, and became independent on CHK1. Conclusion Our results demonstrate that RSR is efficient and rewirable in CSCs thereby constituting a prominent therapeutic target. In particular, we designed dedicated RS-modulating or RSR-targeting strategies for long-term CSC depletion in CRC

Serum epidermal growth factor predicts cognitive functions in early, drug-naive Parkinson's disease patients

Epidermal growth factor (EGF) has been proposed as a candidate biomarker for cognitive impairment in Parkinson's disease (PD). We aimed to assess the relationship between serum EGF and cognitive functions in early, drug-naive PD patients and evaluate the predictive value of EGF on cognitive functions in a 2-year follow-up study. Serum EGF was measured in 65 early, drug-naive PD patients, that underwent a comprehensive neuropsychological battery. Motor symptoms were assessed by means of the Unified Parkinson's Disease Rating Scale, Part III (UPDRS-III). Neuropsychological evaluation was repeated after 2 years. Spearman's rank correlation was used to assess the relationship between serum EGF levels and neuropsychological variables. Linear regression analysis was used to evaluate the relationship between EGF and neuropsychological scores as well as other variables (age, gender, UPDRS-III, levodopa equivalent dose, and type of treatment at follow-up) potentially affecting cognitive performance. Variation over time in cognitive scores was analyzed using repeated-measures ANOVA. At baseline, EGF was the only significant variable associated with performance on semantic fluency (R (2) = 0.131; p = 0.005). EGF levels (p = 0.025), together with UPDRS-III (p = 0.009) and age (p = 0.011), were associated with performance on frontal assessment battery (R (2) = 0.260). At 2-year follow-up, EGF was the only significant variable to predict performance on semantic fluency (R (2) = 0.147; p = 0.025) and color naming task of Stroop color-word test (R (2) = 0.121; p = 0.044). Serum EGF levels are related to frontal and temporal cognitive functions in early, drug-naive PD patients and predict performance on frontal and posterior cognitive functions at 2-year follow-up. EGF is proposed as a potential serum biomarker for early cognitive impairment in PD

Soy isoflavones, inulin, calcium, and vitamin D3 in post-menopausal hot flushes: an observational study.

Purpose of investigation To evaluate the effect of soy isoflavones and inulin (SII) on hot flushes (HF) and quality of life in a clinical setting, the authors conducted an observational study. Materials and methods The authors performed an observational, prospective, multicentric study on women in peri-/post-menopause treated or untreated with a product present on the Italian market, consisting in a mixture of calcium (500 mg), vitamin D3 (300 IU), inulin (3 g) and soy isoflavones (40 mg). Results A total of 135 patients, 75 (55.6%) in the SII group and 60 (44.4%) in the untreated group entered the study. After three months, the mean number of HF declined of 2.8 (SD 3.7) in the SII group and 0.0 in the untreated one. The corresponding values after six months were -3.7 (SD 2.7) in the SII group and -0.9 (SD 5.3) in the control group (p = 0.02). Conclusion This observational trial suggests a possible beneficial effect of a dietary soy supplement containing 40 mg of isoflavone/day plus inulin in the management of menopausal symptoms such as hot flashes

Analysis of the ATR-Chk1 and ATM-Chk2 pathways in male breast cancer revealed the prognostic significance of ATR expression

The ATR-Chk1 and ATM-Chk2 pathways are central in DNA damage repair (DDR) and their over-activation may confer aggressive molecular features, being an adaptive response to endogenous DNA damage and oncogene-induced replication stress. Herein we investigated the ATR-Chk1 and ATM-Chk2 signalings in male breast cancer (MBC). The expression of DDR kinases (pATR, pATM, pChk1, pChk2, and pWee1) and DNA damage markers (pRPA32 and γ-H2AX) was evaluated by immunohistochemistry in 289 MBC samples to assess their association. Survival analyses were carried out in 112 patients. Survival curves were estimated with the Kaplan-Meier method and compared by log-rank test. Cox proportional regression models were generated to identify variables impacting survival outcomes. The expression of pATR conferred poorer survival outcomes (log rank p = 0.013, p = 0.007 and p = 0.010 for overall, 15- and 10-year survival, respectively). Multivariate Cox models of 10-year survival and overall indicated that pATR expression, alone or combined with pChk2, was an independent predictor of adverse outcomes (10-year survival: pATR: HR 2.74, 95% CI: 1.23–6.10; pATR/pChk2: HR 2.92, 95% CI: 1.35–6.33; overall survival: pATR: HR 2.58, 95% CI: 1.20–5.53; pATR/pChk2: HR 2.89, 95% CI: 1.37–6.12). Overall, the ATR/ATM-initiated molecular cascade seems to be active in a fraction of MBC patients and may represent a negative prognostic factor

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations