Modulate Tumor Growth Research Articles

A pilot study of the impact of estrogen signaling on tumor immunity in a syngeneic model of ovarian cancer (213)

A pilot study of the impact of estrogen signaling on tumor immunity in a syngeneic model of ovarian cancer (213)

Ubiquitin-Specific Protease 6 n-Terminal-like Protein (USP6NL) and the Epidermal Growth Factor Receptor (EGFR) Signaling Axis Regulates Ubiquitin-Mediated DNA Repair and Temozolomide-Resistance in Glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant glioma, with a 30–60% epidermal growth factor receptor (EGFR) mutation. This mutation is associated with unrestricted cell growth and increases the possibility of cancer invasion. Patients with EGFR-mutated GBM often develop resistance to the available treatment modalities and higher recurrence rates. The drug resistance observed is associated with multiple genetic or epigenetic factors. The ubiquitin-specific protease 6 N-terminal-like protein (USP6NL) is a GTPase-activating protein that functions as a deubiquitinating enzyme and regulates endocytosis and signal transduction. It is highly expressed in many cancer types and may promote the growth and proliferation of cancer cells. We hypothesized that USP6NL affects GBM chemoresistance and tumorigenesis, and that its inhibition may be a novel therapeutic strategy for GBM treatment. The USP6NL level, together with EGFR expression in human GBM tissue samples and cell lines associated with therapy resistance, tumor growth, and cancer invasion, were investigated. Its pivotal roles and potential mechanism in modulating tumor growth, and the key mechanism associated with therapy resistance of GBM cells, were studied, both in vitro and in vivo. Herein, we found that deubiquitinase USP6NL and growth factor receptor EGFR were strongly associated with the oncogenicity and resistance of GBM, both in vitro and in vivo, toward temozolomide, as evidenced by enhanced migration, invasion, and acquisition of a highly invasive and drug-resistant phenotype by the GBM cells. Furthermore, abrogation of USP6NL reversed the properties of GBM cells and resensitized them toward temozolomide by enhancing autophagy and reducing the DNA damage repair response. Our results provide novel insights into the probable mechanism through which USP6NL/EGFR signaling might suppress the anticancer therapeutic response, induce cancer invasiveness, and facilitate reduced sensitivity to temozolomide treatment in GBM in an autolysosome-dependent manner. Therefore, controlling the USP6NL may offer an alternative, but efficient, therapeutic strategy for targeting and eradicating otherwise resistant and recurrent phenotypes of aggressive GBM cells.

Exosome-Mediated Immunosuppression in Tumor Microenvironments.

Exosomes are membranous structures secreted by nearly all cell types. As critical messengers for intercellular communication, exosomes deliver bioactive cargoes to recipient cells and are involved in multiple physiopathological processes, including immunoregulation. Our pioneering study revealed that cancer cells release programmed death-ligand 1-positive exosomes into the circulation to counter antitumor immunity systemically via T cells. Tumor cell-derived exosomes (TDEs) also play an immunosuppressive role in other immunocytes, including dendritic cells (DCs), macrophages, natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs). Moreover, exosomes secreted by nontumor cells in the tumor microenvironments (TMEs) also exert immunosuppressive effects. This review systematically provides a summary of the immunosuppression induced by exosomes in tumor microenvironments, which modulates tumor growth, invasion, metastasis, and immunotherapeutic resistance. Additionally, therapeutic strategies targeting the molecular mechanism of exosome-mediated tumor development, which may help overcome several obstacles, such as immune tolerance in oncotherapy, are also discussed. Detailed knowledge of the specific functions of exosomes in antitumor immunity may contribute to the development of innovative treatments.

Abstract 1371: Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation

Abstract Immune response is a critical anti-tumor response to overcome tumorigenesis. In line with this, immune checkpoint blockade shows tremendous anti-tumor efficacy by enhancing cytotoxicity of CD8+ cytotoxic T lymphocytes (CTLs). However, cancer cells develop different mechanisms of immune evasion, among which suppression of immune recognition of somatic mutations via tumor antigen is prevalent. In fact, 65% to 90% of the cancers show reduced MHC-I antigen presentation. There is, nonetheless, little achievement toward the therapeutic approaches to restore antigen presentation on tumor cells. Colorectal cancer (CRC), the second deadliest cancer in U.S., shows increasing incidence and mortality rates over the past years. Standard conventional CRC treatments, surgery, chemotherapy and radiotherapy, often lead to many side effects due to their non-specificity and cytotoxicity. While immunotherapy is very promising, only a small population of CRC patients respond to the current immune checkpoint inhibitors. In this study, our objective was to harness a novel immunological approach to target CRC tumors. We developed a bioinformatics tool to create a pool of genes whose expression negatively correlates with cytotoxicity of CD8+ CTLs in the tumor microenvironment of CRC patients. Given the central role of antigen presentation in activating CD8+ CTLs, we further screened these genes based on the scope of their effects on antigen presentation. To this end, we developed stable clones of OVA expressing MC38 cells (MC38OVA) where OVA-derived peptide, SIINFEKL, will be bound to MHC-I complex for cell surface presentation. 4 genes with highest capacity to alter MHC-I cell surface levels were identified. Our consequent in vivo experiments and in vitro cytotoxicity assays identified that “Rho-GEF” gene was the top actionable target with striking suppressive effects on tumor growth. TCGA database analysis showed Rho-GEF is upregulated in 22% of CRC patients accompanied by a poor prognosis. Rho-GEF knockdown (KD) significantly inhibited tumor growth in immunocompetent mice whereas it only showed minor effects on immunocompromised mice, suggesting that the Rho-GEF may modulate tumor growth by regulating the interaction of tumor cells with non-tumor cells such as immune cells. Consistent with the major contribution of antigen presentation in promoting CD8+ CTLs activity, TNFα, IFNγ, and GZMB levels in CD8+ T cells isolated from Rho-GEF KD MC38-derived tumors showed a significant increase compared to the wild type tumors. Taken together, we introduced Rho-GEF as a potential clinical target involved in CRC immune evasion by modulating antigen presentation and CD8+ CTLs anti-tumor responses. Citation Format: Haniyeh Eyvani, Xinna Zhang. Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation [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 1371.

Unconventional Protein Secretion in Brain Tumors Biology: Enlightening the Mechanisms for Tumor Survival and Progression.

Non-canonical secretion pathways, collectively known as unconventional protein secretion (UPS), are alternative secretory mechanisms usually associated with stress-inducing conditions. UPS allows proteins that lack a signal peptide to be secreted, avoiding the conventional endoplasmic reticulum-Golgi complex secretory pathway. Molecules that generally rely on the canonical pathway to be secreted may also use the Golgi bypass, one of the unconventional routes, to reach the extracellular space. UPS studies have been increasingly growing in the literature, including its implication in the biology of several diseases. Intercellular communication between brain tumor cells and the tumor microenvironment is orchestrated by various molecules, including canonical and non-canonical secreted proteins that modulate tumor growth, proliferation, and invasion. Adult brain tumors such as gliomas, which are aggressive and fatal cancers with a dismal prognosis, could exploit UPS mechanisms to communicate with their microenvironment. Herein, we provide functional insights into the UPS machinery in the context of tumor biology, with a particular focus on the secreted proteins by alternative routes as key regulators in the maintenance of brain tumors.

Abstract 3530: Microbial-Interleukin 17 receptor A (IL-17RA) signaling axis modulates tumor growth and microenvironment

Abstract Microbiota, both within the gut and the tumors, has emerged as a significant player influencing tumor growth and responses to therapies. Recent evidence links microbiota and pancreatic ductal adenocarcinoma (PDAC), an aggressive cancer surrounded by a highly immuno-suppressive tumor microenvironment which limits efficacy of most available therapies. The immunosuppressive tumor microenvironment of PDAC is partially facilitated by a proinflammatory cytokine Interleukin 17 (IL-17). IL-17 can be stimulated by intestinal commensal bacteria under normal physiological conditions. It is critical for microbial defense as well as immunopathology. Both IL-17 neutralization and antibiotics reduce murine PDAC growth. While the vital role of microbiota in affecting tumor immunity has been identified, there is still a gap of knowledge as to how microbes may regulate pro-tumorigenic IL-17 signaling. We wanted to investigate the systemic and local role of IL-17 in regulating the microbial-tumor immune axis. For this purpose, we genetically deleted the IL-17 receptor A (IL-17RA) in different compartments and evaluated tumor growth. We found that IL-17RA signaling was important for maintaining microbial homeostasis and its disruption resulted in differential tumor growth. Absence of IL-17RA signaling in the gut lead to local inflammation as well as systemic immune effects. Our data suggests that modulation of IL-17 signaling could serve as a therapeutic intervention to alter microbial mediated tumor effects. The significance of these findings may extend to other cancers as well. Citation Format: Vidhi Chandra, Yu Zhang, Olivereen Le Roux, Joseph Petrosino, Jay Kolls, Florencia McAllister. Microbial-Interleukin 17 receptor A (IL-17RA) signaling axis modulates tumor growth and microenvironment [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 3530.

Prognostic value of hematologic parameters in patients with muscle invasive bladder cancer.

e16502 Background: Muscle invasive bladder cancers (MIBC) tend to have an increased risk of progression and recurrence despite optimal management. Since various blood cells affect tumor microenvironments, modulate tumor growth and treatment responses, we examined the impact of Platelet/Lymphocyte ratio (PLR) and systemic inflammation response index [(SIRI) = N×M/L), N- Neutrophil, M-monocyte, and L-Lymphocyte] at diagnosis of MIBC patients on overall prognosis. Methods: We performed a retrospective analysis of all muscle invasive bladder cancer patients treated at our institute from January 2015 to December 2020. Patients with Stage I and IV disease were excluded. Descriptive statistics were obtained for entire cohort. The optimal cutoff points for PLR and SIRI were accessed by the receiver operating characteristic curve (ROC) based on overall survival (OS) and Progression free survival (PFS). Kaplan-Meier curve was used to compare the OS and the PFS between the groups. Univariate and multivariate Cox regression analyses were used to evaluate the impact of PLR and SIRI on the OS. Results: In our cohort of 112 patients, median age at diagnosis was 65 years (95% CI: 62-68). Patients were predominantly males (74.1%), Caucasians (88.3%), smokers (71.4%), and 59.8% had a BMI above 24.9. The median duration of follow up was 18.7 months (mo). The median OS was 28.5 mo (95% CI: 19.8-55.4) and median PFS was 16.7 mo (95% CI: 11.7-30.0). 50 patients (44.6%) were stage II and 62 patients (55.4%) were Stage III. 34 patients (30.4%) received chemotherapy in neoadjuvant or adjuvant setting, 6 patients (5.4%) received radiotherapy, and majority of patients (total 75; 67.0%) were treated with surgical intervention. The optimal cutoff points on OS for PLR and SIRI were 304.04 and 1.83 respectively. On univariate analysis, SIRI did not affect OS (HR: 1.26, 95% CI: 0.73-2.19, p = 0.406). Patients with high PLR had worse OS on both univariate (HR: 1.003, 95% CI: 1.001-1.005, p = 0.003) and multivariate (HR: 1.002, 95% CI: 1.000-1.004, p = 0.028) analyses. Nodal staging, radiation treatment, and baseline hemoglobin level were other statistically significant factors associated with OS. Conclusions: Although the standard TNM staging system is helpful in predicting tumor behavior, there is marked heterogeneity in responses to treatment for MIBC. Our study suggests that PLR at diagnosis can be a novel prognostic marker for patients with MIBC.

Sensory Neurotransmitter Calcitonin Gene-Related Peptide Modulates Tumor Growth and Lymphocyte Infiltration in Oral Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma are highly innervated by peripheral sensory neurons. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from sensory neurons innervating cancer is linked to tumorigenesis. CGRP-immunoreactive nerve presence comprised 9.53±1.9% of total nerve area across 11 HNSCC patients. A syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (CGRPKO ) are used to investigate the impact of CGRP signaling on tumor growth and the associated immune response in vivo. In tumor-bearing CGRPKO mice, there is a significant reduction in tumor size over time compared to wildtype mice using two different mouse oral cancer cell lines. Furthermore, tumor tissue from CGRPKO mice had a significant increase in tumor-infiltrating CD4+ T cells, cytotoxic CD8+ T cells, and NK1.1+ NK cells compared to wildtype. Fluorescent-activated cell sorting and real-time qPCR are used to confirm that CD4+ T cells are isolated from tumor-bearing wildtype mice containing a high expression of Ramp1 compared to sham mice. These data suggest that sensory neurotransmitter CGRP may modulate oral cancer progression via tumor immunosurveillance. Understanding the relationship between sensory neurons and cancer will aid in repurposing clinically available nervous system drugs for the treatment of cancer.

Demystifying the Relationship Between Metformin, AMPK, and Doxorubicin Cardiotoxicity.

Doxorubicin (DOX) is an extremely effective and wide-spectrum anticancer drug, but its long-term use can lead to heart failure, which presents a serious problem to millions of cancer survivors who have been treated with DOX. Thus, identifying agents that can reduce DOX cardiotoxicity and concurrently enhance its antitumor efficacy would be of great clinical value. In this respect, the classical antidiabetic drug metformin (MET) has stood out, appearing to have both antitumor and cardioprotective properties. MET is proposed to achieve these beneficial effects through the activation of AMP-activated protein kinase (AMPK), an essential regulator of mitochondrial homeostasis and energy metabolism. AMPK itself has been shown to protect the heart and modulate tumor growth under certain conditions. However, the role and mechanism of the hypothesized MET-AMPK axis in DOX cardiotoxicity and antitumor efficacy remain to be firmly established by in vivo studies using tumor-bearing animal models and large-scale prospective clinical trials. This review summarizes currently available literature for or against a role of AMPK in MET-mediated protection against DOX cardiotoxicity. It also highlights the emerging evidence suggesting distinct roles of the AMPK subunit isoforms in mediating the functions of unique AMPK holoenzymes composed of different combinations of isoforms. Moreover, the review provides a perspective regarding future studies that may help fully elucidate the relationship between MET, AMPK and DOX cardiotoxicity.

Programmed death ligand 1 gene silencing in murine glioma models reveals cell line-specific modulation of tumor growth in vivo.

BackgroundGlioblastoma is the most common brain tumor in adults and virtually incurable. Therefore, new therapeutic strategies are urgently needed. Immune checkpoint inhibition has not shown activity in various phase III trials and intra- as well as intertumoral expression of programmed death ligand 1 (PD-L1) varies in glioblastoma.MethodsWe abrogated constitutive PD-L1 gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo.ResultsA heterogeneous expression of Pdl1 mRNA and PD-L1 protein was detected in the glioma cell panel in vitro and in vivo. PD-L1, but not PD-L2, was inducible by interferon β and γ. Co-culture with splenocytes induced PD-L1 expression in GL-261 and SMA-560, but not in CT-2A cells, in an interferon γ-dependent manner. Conversely, Pdl1 gene silencing conferred a survival benefit in CT-2A, but not in the other 2 models. Accordingly, PD-L1 antibody prolonged survival in CT-2A glioma-bearing mice. This activity required PD-L1 expression on tumor rather than host cells, and the survival gain mediated by PD-L1 loss was reproduced in immune-deficient RAG−/− mice.ConclusionsPD-L1 is expressed and interferon-inducible in murine glioma cell lines. PD-L1 has model-specific roles for tumor growth. Future studies need to determine which subset of glioblastoma patients may benefit from PD-L1 antagonism as part of a multimodality therapeutic approach to glioblastoma.

Cisplatin Uptake in Macrophage Subtypes at the Single-Cell Level by LA-ICP-TOFMS Imaging.

A high-throughput laser ablation–inductively coupled plasma–time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.

Innate Immunity and Cancer Pathophysiology.

Chronic inflammation increases the risk of several cancers, including gastric, colon, and hepatic cancers. Conversely, tumors, similar to tissue injury, trigger an inflammatory response coordinated by the innate immune system. Cellular and molecular mediators of inflammation modulate tumor growth directly and by influencing the adaptive immune response. Depending on the balance of immune cell types and signals within the tumor microenvironment, inflammation can support or restrain the tumor. Adding to the complexity, research from the past two decades has revealed that innate immune cells are highly heterogeneous and plastic, with variable phenotypes depending on tumor type, stage, and treatment. The field is now on the cusp of being able to harness this wealth of data to (a) classify tumors on the basis of their immune makeup, with implications for prognosis, treatment choice, and clinical outcome, and (b) design therapeutic strategies that activate antitumor immune responses by targeting innate immune cells.

CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells

Introduction:Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized.Methods:CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining.Results:To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p <0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p<0.005 and 2-fold decrease for H929, p<0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p <0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI).Conclusion:We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. DisclosuresGhiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.

Immunomodulatory Effects of Jacalin, a Dietary Plant Lectin on the Peripheral Blood Mononuclear Cells (PBMCs).

The tumor microenvironment that refers to the tumor's surroundings is a key modulator of tumor growth and invasion. The tumor-derived signals are known to downregulate the anti-tumor effects of the effector cells present in the TME. Thus, the cross-talk between the tumor cells with the surrounding immune cells helps in evading the tumor surveillance as well as aiding in tumor growth and proliferation. Hence, knowledge regarding the effects of drugs/compound on the tumor-stromal interactions is gaining importance. In the present study, the effects of jacalin, a dietary lectin on the proliferation and cytokine production of peripheral blood mononuclear cells (PBMCs), are investigated. Jacalin was shown to act as a mitogen of PBMCs, the key cytokine secreting immune cells. Also, jacalin initially induced increased mRNA expression of pro-inflammatory cytokine IFN-γ; however, prolonged stimulation of PBMCs resulted in increased expression of anti-inflammatory cytokine, mainly TGF-β. Furthermore, 6h jacalin prestimulated PBMCs (Jac-PBMCs) were shown to inhibit HeLa cell proliferation while 24h Jac-PBMCs were found to favor tumor growth. Thus, it may be postulated that while jacalin initially polarizes the PBMCs to hinder the tumor growth, after a stipulated time point, interaction of jacalin with PBMCs can lead to an immunosuppressive TME that may probably assist in tumor growth and progression.

Reticulon 3-mediated Chk2/p53 activation suppresses hepatocellular carcinogenesis and is blocked by hepatitis B virus

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining...

Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology.

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation

Introduction: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2′-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). Methods: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. Results: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/β-catenin. Conclusions: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Profiling Cancer-Associated Fibroblasts in Melanoma.

Solid tumors are complex systems characterized by dynamic interactions between neoplastic cells, non-tumoral cells, and extracellular components. Among all the stromal cells that populate tumor microenvironment, fibroblasts are the most abundant elements and are critically involved in disease progression. Cancer-associated fibroblasts (CAFs) have pleiotropic functions in tumor growth and extracellular matrix remodeling implicated in local invasion and distant metastasis. CAFs additionally participate in the inflammatory response of the tumor site by releasing a variety of chemokines and cytokines. It is becoming clear that understanding the dynamic, mutual melanoma–fibroblast relationship would enable treatment options to be amplified. To better characterize melanoma-associated fibroblasts, here we analyzed low-passage primary CAFs derived from advanced-stage primary skin melanomas, focusing on the immuno-phenotype. Furthermore, we assessed the expression of several CAF markers and the production of growth factors. To deepen the study of CAF–melanoma cell crosstalk, we employed CAF-derived supernatants and trans-well co-culture systems to evaluate the influences of CAFs on (i) the motogenic ability of melanoma cells, (ii) the chemotherapy-induced cytotoxicity, and (iii) the release of mediators active in modulating tumor growth and spread.

Natural Killer Cells Recruitment in Oncolytic Virotherapy: A Mathematical Model.

In this paper, we investigate how natural killer (NK) cell recruitment to the tumor microenvironment (TME) affects oncolytic virotherapy. NK cells play a major role against viral infections. They are, however, known to induce early viral clearance of oncolytic viruses, which hinders the overall efficacy of oncolytic virotherapy. Here, we formulate and analyze a simple mathematical model of the dynamics of the tumor, OV and NK cells using currently available preclinical information. The aim of this study is to characterize conditions under which the synergistic balance between OV-induced NK responses and required viral cytopathicity may or may not result in a successful treatment. In this study, we found that NK cell recruitment to the TME must take place neither too early nor too late in the course of OV infection so that treatment will be successful. NK cell responses are most influential at either early (partly because of rapid response of NK cells to viral infections or antigens) or later (partly because of antitumoral ability of NK cells) stages of oncolytic virotherapy. The model also predicts that: (a) an NK cell response augments oncolytic virotherapy only if viral cytopathicity is weak; (b) the recruitment of NK cells modulates tumor growth; and (c) the depletion of activated NK cells within the TME enhances the probability of tumor escape in oncolytic virotherapy. Taken together, our model results demonstrate that OV infection is crucial, not just to cytoreduce tumor burden, but also to induce the stronger NK cell response necessary to achieve complete or at least partial tumor remission. Furthermore, our modeling framework supports combination therapies involving NK cells and OV which are currently used in oncolytic immunovirotherapy to treat several cancer types.

Nuclear ErbB-2-Induced Transcriptome Drives Triple Negative Breast Cancer Growth

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS(HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.