DEVELOPMENT AND CHARACTERIZATION OF NEW pan-SIGMA RECEPTOR MODULATORS: CHEMICAL, BIOLOGICAL AND PHARMACOLOGICAL STUDIES FOR PRECLINICAL VALIDATION OF NEW THERAPEUTIC TARGETS

Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB.Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB

The promise of liquid biopsy in cancer: a clinical perspective

The clinical utility of liquid biopsy in cancer treatment will increase as circulating tumor cells (CTCs) analysis move from the enumeration to the real-time measurement of tumor characteristics. Intratumor heterogeneity is becoming increasingly recognized as a major drawback to the shift to personalized medicine. Spatial and temporal heterogeneity might be reflected by the serial assessment of CTCs. Indeed, the developing technologies for CTCs analysis now allow digital genomic and next-generation sequencing approaches, able to differentiate molecular subtypes of the disease and to monitor genetic variation over time. The liquid biopsy of cancer might offer a real-time assessment of tumor biology, providing the opportunity to serially evaluate patients most likely to benefit from targeted drugs based on a dynamic characterization of the disease at the molecular level. Although hurdles remain before liquid biopsy is seen in routine clinical practice, the information derived from CTCs may facilitate the real-time identification of actionable mutations in cancer leading the way toward personalized medicine

Core stability in young female dancers

Core has been described as a corset stabilizing the trunk area, bordered by the diaphragm upwardly, the abdominal muscular complex anterior-laterally, the spinal and gluteal muscles posteriorly and by the pelvic floor and the muscles of the pelvic girdle inferiorly (1). Core Stability (CS) is intended as a neuromuscular skill of trunk control, assisted by passive (ligaments) and active (muscles) elements. The effects of CS training on the strength, endurance and balance of young female dancers were measured. Thirty three young women participated (22 amateur dancers; 11 sedentary people; age 21±5.4yr) were asked to perform endurance (2), strength, and balance (3) tests (Session I) that were repeated after 10 weeks (Session II). During this period, 11 dancers (experimental group, EG) were randomly selected to attend specific training for CS in addition to traditional dancing exercises; 11 dancers only performed regular dance training (dance group, DG); the sedentary people did not carry out any exercise (control group, CG). Within each group and session, descriptive statistics of test performances were computed. Differences between groups and sessions were assessed, setting the level of statistical significance at 5% (p≤0.05) for all comparisons. In both sessions, EG and DG were stronger and more resistant than CG and demonstrated better balance in two balance tests (

Prognostic role of circulating tumor cell trajectories in metastatic colorectal cancer

Abstract: Background: A large amount of evidence from clinical studies has demonstrated that circulating tumor cells are strong predictors of outcomes in many cancers. However, the clinical significance of CTC enumeration in metastatic colorectal cancer is still questioned. The aim of this study was to evaluate the clinical value of CTC dynamics in mCRC patients receiving first-line treatments. Materials and methods: Serial CTC data from 218 patients were used to identify CTC trajectory patterns during the course of treatment. CTCs were evaluated at baseline, at a first-time point check and at the radiological progression of the disease. CTC dynamics were correlated with clinical endpoints. Results: Using a cut-off of ≥1 CTC/7.5 mL, four prognostic trajectories were outlined. The best prognosis was obtained for patients with no evidence of CTCs at any timepoints, with a significant difference compared to all other groups. Lower PFS and OS were recognized in group 4 (CTCs always positive) at 7 and 16 months, respectively. Conclusions: We confirmed the clinical value of CTC positivity, even with only one cell detected. CTC trajectories are better prognostic indicators than CTC enumeration at baseline. The reported prognostic groups might help to improve risk stratification, providing potential biomarkers to monitor first-line treatments

Genomic landscape and survival analysis of ctDNA “neo-RAS wild-type” patients with originally RAS mutant metastatic colorectal cancer

Background: The term “neo-RAS wild-type” refers to the switch to RAS wild-type disease in plasma circulating tumor DNA (ctDNA) from originally RAS mutant colorectal cancers. Consistently, the hypothesis to re-determine RAS mutational status in ctDNA at disease progression in RAS mutant mCRC opened to a new perspective for clinically-based selection of patients to be treated with EGFR inhibitors. Currently, the genomic landscape of “neo-RAS wild-type” is unknown. This is a prospective study aimed to investigate clinical and genomic features associated with RAS mutation clearance in a large cohort of RAS mutant mCRC patients who converted to RAS wild- type in liquid biopsy at failure of first-line treatments. Secondary aim was to investigate the long term prognostic significance of “true neo-RAS wild- type”. Patients and methods: 70 patients with stage IV RAS mutant colorectal cancer were prospectively enrolled. Plasma samples were collected at progression from first-line treatment. RAS/BRAF mutations in plasma were assessed by RT-PCR. In RAS/BRAF wild-type samples, ctDNA was used to generate libraries using a 17 genes panel whose alteration has clinical relevance. To investigate the prognostic significance of RAS mutation clearance, test curves for PFS and OS were represented by Kaplan-Meier estimator plot and Log-rank test. Results: The most commonly detected actionable mutations in “neo-RAS wild-type” were: PIK3CA (35.7%); RET (11.9%); IDH1 (9.5%); KIT (7%); EGFR (7%); MET (4.7%); ERBB2 (4.7%); FGFR3 (4.7%). Both OS and post-progression survival were longer in patients with “neo-RAS wild-type” compared to those who remained RAS mutant (p<0.001 for both). Conclusions: De-novo-targetable mutations occured in a large percentage of “neo-RAS wild-type”, being PIK3CA the most commonly detected. RAS mutation clearance in ctDNA is associated with long- term improvement of overall survival

The role of opioids in cancer response to immunotherapy

BACKGROUND: The response to immunotherapy can be impaired by several factors including external intervention such as drug interactions with immune system. We aimed to examine the immunomodulatory action of opioids, since immune cells express opioid receptors able to negatively influence their activities.METHODS: This observational, multicenter, retrospective study, recruited patients with different metastatic solid tumors, who have received immunotherapy between September 2014 and September 2019. Immunotherapy was administered according to the standard schedule approved for each primary tumor and line of treatment. The concomitant intake of antibiotics, antifungals, corticosteroids and opioids were evaluated in all included patients. The relationship between tumor response to immunotherapy and the oncological outcomes were evaluated. A multivariate Cox-proportional hazard model was used to identify independent prognostic factors for survival.RESULTS: One hundred ninety-three patients were recruited. Overall, progression-free survival (PFS) and overall survival (OS) were significantly shorter in those patients taking opioids than in those who didn't (median PFS, 3months vs. 19months, HR 1.70, 95% CI 1.37-2.09, p<0.0001; median OS, 4months vs. 35months, HR 1.60, 95% CI 1.26-2.02, p<0.0001). In addition, PFS and OS were significantly impaired in those patients taking corticosteroids, antibiotics or antifungals, in those patients with an ECOG PS≥1 and in patients with a high tumor burden. Using the multivariate analyses, opioids and ECOG PS were independent prognostic factors for PFS, whereas only ECOG PS resulted to be an independent prognostic factor for OS, with trend toward significance for opioids as well as tumor burden.DISCUSSION: Our study suggests that the concomitant administration of drugs as well as some clinical features could negatively predict the outcomes of cancer patients receiving immunotherapy. In particular, opioids use during immunotherapy is associated with early progression, potentially representing a predictive factor for PFS and negatively influencing OS as well.CONCLUSIONS: A possible negative drug interaction able to impair the immune response to anti-PD-1/PD-L1 agents has been highlighted. Our findings suggest the need to further explore the impact of opioids on immune system modulation and their role in restoring the response to immunotherapy treatment, thereby improving patients' outcomes

Sigma Receptors as Endoplasmic Reticulum Stress “Gatekeepers” and their Modulators as Emerging New Weapons in the Fight Against Cancer

Despite the interest aroused by sigma receptors (SRs) in the area of oncology, their role in tumor biology remains enigmatic. The predominant subcellular localization and main site of activity of SRs are the endoplasmic reticulum (ER). Current literature data, including recent findings on the sigma 2 receptor subtype (S2R) identity, suggest that SRs may play a role as ER stress gatekeepers. Although SR endogenous ligands are still unknown, a wide series of structurally unrelated compounds able to bind SRs have been identified. Currently, the identification of novel antiproliferative molecules acting via SR interaction is a challenging task for both academia and industry, as shown by the fact that novel anticancer drugs targeting SRs are in the preclinical-stage pipeline of pharmaceutical companies (i.e., Anavex Corp. and Accuronix). So far, no clinically available anticancer drugs targeting SRs are still available. The present review focuses literature advancements and provides a state-of-the-art overview of SRs, with emphasis on their involvement in cancer biology and on the role of SR modulators as anticancer agents. Findings from preclinical studies on novel anticancer drugs targeting SRs are presented in brief

Inclusion of Platinum Agents in Neoadjuvant Chemotherapy Regimens for Triple-Negative Breast Cancer Patients: Development of GRADE (Grades of Recommendation, Assessment, Development and Evaluation) Recommendation by the Italian Association of Medical Oncology (AIOM)

In the absence of identified therapeutic targets, chemotherapy is the main systemic treatment option for triple-negative breast cancer (TNBC). The achievement of a pathological complete response (pCR) after neoadjuvant chemotherapy leads to good outcome, whereas patients not achieving a pCR are at high risk of relapse. Various trials have evaluated the inclusion of platinum in neoadjuvant chemotherapy regimens for TNBC, leading to non-univocal results. The panel of the Italian Association of Medical Oncology (AIOM) Guidelines on Breast Cancer developed a clinical recommendation on the addition of platinum to anthracycline/taxane-based neoadjuvant chemotherapy for TNBC by using the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology and the Evidence to Decision framework (EtD). Five studies were eligible. The panel identified the following outcomes of benefit: pCR (critical), disease/event-free survival (DFS/EFS, critical), and overall survival (OS, critical). The panel identified febrile neutropenia (critical), serious adverse events (critical), anemia grade 3-4 (important), thrombocytopenia grade 3-4 (important) as outcomes of harms. The probability of pCR was higher in the platinum-based chemotherapy group versus control group (RR = 1.45, 95%CI 1.28-1.64); however, no impact on long-term outcome was observed. Neoadjuvant treatment regimens containing platinum resulted in a non-significant increase in the risk of febrile neutropenia and in a significant increase in the risk serious adverse events, G3-G4 anemia and G3-G4 thrombocytopenia: 11.3% versus 0.8%, RR = 15.66 (95%CI 6.38-38.44). The panel judged uncertain/favorable the benefit/harms balance. The panel's final recommendation was conditional in favor of the inclusion of platinum in anthracycline/taxane-based neoadjuvant regimens for TNBC

TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non small cell lung cancer

Background Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms. Methods We investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay. Results We demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization. Conclusions In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors