Thymoquinone, a Novel Multi-Strike Inhibitor of Pro-Tumorigenic Breast Cancer (BC) Markers: CALR, NLRP3 Pathway and sPD-L1 in PBMCs of HR+ and TNBC Patients.

Breast cancer (BC) is not only a mass of malignant cells but also a systemic inflammatory disease. BC pro-tumorigenic inflammation has been shown to promote immune evasion and provoke BC progression. The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is activated when pattern recognition receptors (PRRs) sense danger signals such as calreticulin (CALR) from damaged/dying cells, leading to the secretion of interleukin-1ß (IL-1ß). CALR is a novel BC biological marker, and its high levels are associated with advanced tumors. NLRP3 expression is strongly correlated with an elevated proliferative index Ki67, BC progression, metastasis, and recurrence in patients with hormone receptor-positive (HR+) and triple-negative BC (TNBC). Tumor-associated macrophages (TAMs) secrete high levels of IL-1ß promoting endocrine resistance in HR+ BC. Recently, an immunosuppressive soluble form of programmed death ligand 1 (sPD-L1) has been identified as a novel prognostic biomarker in triple-negative breast cancer (TNBC) patients. Interestingly, IL-1ß induces sPD-L1 release. BC Patients with elevated IL-1ß and sPD-L1 levels show significantly short progression-free survival. For the first time, this study aims to investigate the inhibitory impact of thymoquinone (TQ) on CALR, the NLRP3 pathway and sPD-L1 in HR+ and TNBC. Blood samples were collected from 45 patients with BC. The effect of differing TQ concentrations for different durations on the expression of CALR, NLRP3 complex components and IL-1ß as well as the protein levels of sPD-L1 and IL-1ß were investigated in the peripheral blood mononuclear cells (PBMCs) and TAMs of TNBC and HR+ BC patients, respectively. The findings showed that TQ significantly downregulated the expression of CALR, NLRP3 components and IL-1ß together with the protein levels of secreted IL-1ß and sPD-L1. The current findings demonstrated novel immunomodulatory effects of TQ, highlighting its potential role not only as an excellent adjuvant but also as a possible immunotherapeutic agent in HR+ and TNBC patients.

A cutting-edge immunomodulatory interlinkage between HOTAIR and MALAT1 in tumor-associated macrophages in breast cancer: A personalized immunotherapeutic approach.

Breast cancer (BC) is one of the most common cancers, accounting for 2.3 million cases worldwide. BC can be molecularly subclassified into luminal A, luminal B HER2-, luminal B HER2+, HER2+, and triple-negative breast cancer (TNBC). These molecular subtypes differ in their prognosis and treatment strategies; thus, understanding the tumor microenvironment (TME) of BC could lead to new potential treatment strategies. The TME hosts a population of cells that act as antitumorigenic such as tumor-associated eosinophils or pro-tumorigenic such as cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs), monocytic-derived populations such as MDSCs, or most importantly "tumor-associated macrophages (TAMs)," which are derived from CD14+ monocytes. TAMs are reported to have the pro-inflammatory phenotype M1, which is found only in the very early stages of tumor and is not correlated with progression; however, the M2 phenotype is anti-inflammatory that is correlated with tumor progression and metastasis. The current study focused on controlling the anti-inflammatory activity in TAMs of hormonal, HER2+, and TNBC by epigenetic fine-tuning of two immunomodulatory proteins, namely, CD80 and mesothelin (MSLN), which are known to be overexpressed in BC with pro-tumorigenic activity. Long non-coding RNAs are crucial key players in tumor progression whether acting as oncogenic or tumor suppressors. We focused on the regulatory role of MALAT1 and HOTAIR lncRNAs and their role in controlling the tumorigenic activity of TAMs. This study observed the impact of manipulation of MALAT1 and HOTAIR on the expression of both CD80 and MSLN in TAMs of BC. Moreover, we analyzed the interlinkage between HOTAIR and MALAT1 as regulators to one another in TAMs of BC. The current study reported an upstream regulatory effect of HOTAIR on MALAT1. Moreover, our results showed a promising use of MALAT1 and HOTAIR in regulating oncogenic immune-modulatory proteins MSLN and CD80 in TAMs of HER2+ and TNBC. The downregulation of MALAT1 and HOTAIR resulted in the upregulation of CD80 and MSLN, which indicates that they might have a cell-specific activity in TAMs. These data shed light on novel key players affecting the anti-inflammatory activity of TAMs as a possible therapeutic target in HER2+ and TNBC.

CXCR2 Small-Molecule Antagonist Combats Chemoresistance and Enhances Immunotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer as the absence of cell surface receptors renders it more difficult to be therapeutically targeted. Chemokine receptor 2 (CXCR2) has been suggested not only to promote therapy resistance and suppress immunotherapy but it also to possess a positive cross-talk with the multifunctional cytokine transforming growth factor beta (TGF-ß). Here, we showed that CXCR2 and TGF-ß signaling were both upregulated in human TNBC biopsies. CXCR2 inhibition abrogated doxorubicin-mediated TGF-ß upregulation in 3D in vitro TNBC coculture with PBMCs and eliminated drug resistance in TNBC mammospheres, suggesting a vital role for CXCR2 in TNBC doxorubicin-resistance via TGF-ß signaling regulation. Moreover, CXCR2 inhibition improved the efficacy of the immunotherapeutic drug "atezolizumab" where the combined inhibition of CXCR2 and PDL1 in TNBC in vitro coculture showed an additive effect in cytotoxicity. Altogether, the current study suggests CXCR2 inhibitors as a promising approach to improve TNBC treatment if used in combination with chemotherapy and/or immunotherapy.

NEAT1: Culprit lncRNA linking PIG-C, MSLN, and CD80 in triple-negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women. Despite the effectiveness of conventional therapies, they cause detrimental side effects. Glycosyl-Phosphatidyl-Inositol (GPI) pathway is a conserved pathway that culminates in the generation of GPI anchored proteins (GPI-AP). Phosphatidyl-Inositol-Glycan Biosynthesis Class C (PIG-C) is the first step in GPI pathway and upon its overexpression, Mesothelin (MSLN); an oncogenic GPI-AP, expression is induced. Therefore, blocking GPI pathway is a potential therapy through which multiple pathways can be rectified. Recombinant GPI-CD80 proved to be a potent immunostimulatory protein and currently being evaluated as tumor vaccine. In fact, CD80 is a unique immunomodulator that binds to CD28, CTLA-4 and PD-L1. Furthermore, research advancement showed that non-coding RNAs (ncRNAs) are key epigenetic modulators. Therefore, epigenetic tuning of GPI-APs remains an unexplored area. This study aims at investigating the potential role of ncRNAs in regulating MSLN, PIG-C and CD80 in BC. METHODS: Potential ncRNAs were filtered by bioinformatics algorithms. MDA-MB-231 cells were transfected with RNA oligonucleotides. Surface CD80 and MSLN were assessed by FACS and immunofluorescence. Gene expression was tested by q-PCR. RESULTS: PIG-C gene was overexpressed in TNBC and its manipulation altered MSLN surface level. Aligning with bioinformatics analysis, miR-2355 manipulated PIG-C and MSLN expression, while miR-455 manipulated CD80 expression. NEAT1 sponged both miRNAs. Paradoxically, NEAT1 lowered PIG-C gene expression while increased MSLN gene expression. CONCLUSION: This study unravels novel immunotherapeutic targets for TNBC. NEAT1 is potential immunomodulator by sponging several miRNAs. Finally, this study highlights GPI pathway applications, therefore integrating epigenetics, post-translational modifications and immunomodulation.

Oleuropin controls miR-194/XIST/PD-L1 loop in triple negative breast cancer: New role of nutri-epigenetics in immune-oncology.

Oleuropein, the main secoiridoid glucoside found in Olea europaea L., has attracted scientific community as a potential anticancer agent. Immunotherapy and RNA interference revolutionized cancer treatment. Success of PD-L1/PD-1 antibodies encouraged the investigation of PD-1/PD-L1 regulation by non-coding RNAs. This study aimed to verify the cytotoxic effect of oleuropein on MDA-MB-231 cell line and to unravel novel ceRNA interaction between miR-194-5p and XIST in breast cancer and their immunomodulatory effect on PD-L1 expression to propose a promising prophylactic and preventive role of Oleuropin in diet. For the first time, miR-194/Lnc-RNA XIST/PD-L1 triad was investigated in breast cancer, where miR-194 and PD-L1 levels were significantly upregulated in 21 BC-biopsies, yet XIST was downregulated. Ectopic expression of miR-194 enhanced cell function and viability with concomitant increase in PD-L1 expression yet XIST expression decreased, in contrast to miR-194 antagomirs that yielded opposite results. XIST knock-out elevated miR194-5p and PD-L1 levels. miR-194-5p mimics and XIST siRNAs co-transfection induced PD-L1 expression, while miR-194-5p mimics and TSIX siRNAs co-transfection showed opposite effect. Oleuropein showed anti-carcinogenic impact by decreasing miR-194 and PD-L1 levels while increasing XIST level. In conclusion, our study highlighted novel ceRNA interaction controlling PD-L1 expression in BC. Oleuropein is a promising nutraceutical for cancer therapy. Therefore, oleuropin represents a new nutri-epigenetic in immune-oncology that controls miR-194/XIST/PD-L1 loop in triple negative breast cancer.

MALAT-1: LncRNA ruling miR-182/PIG-C/mesothelin triad in triple negative breast cancer.

Breast cancer (BC) remains a major health problem, despite the remarkable advances in cancer research setting. BC is the most common cancer affecting women worldwide. In the context of triple negative breast cancer (TNBC) treatment, major obstacles include late diagnoses and detrimental side effects of chemotherapy and radiotherapy. Research effort was rewarded with the discovery of mesothelin (MSLN), an oncogenic Glycosyl-Phosphatidyl-Inositol (GPI) anchored protein, over-expressed in TNBC. GPI pathway is a post-translational modification that attaches proteins to cellular membrane. MSLN targeted therapy succeeded in early clinical trials, nevertheless, to date, the epigenetic regulation of MSLN and GPI pathway by non-coding RNAs (nc-RNAs) in BC remains an untouched area. Accordingly, our aim is to investigate-for the first time- the impact of simultaneous targeting of MSLN and its associated GPI pathway member, PIG-C, by non-coding-RNAs. Expression profiling of PIG-C, MSLN in BC was performed. Using bioinformatics tools, MALAT-1 and miR-182 were found to target MSLN and PIG-C. MDA-MB-231 cells were transfected with synthetic nc-RNAs. Expression profiling of MSLN, miR-182 and MALAT-1 showed a dramatic over-expression in BC samples. MiR-182 ectopic expression and MALAT-1 silencing increased MSLN and PIG-C transcript levels. However, miR-182 inhibition and miR-182/si-MALAT-1 co-transfection lowered MSLN and PIG-C levels. Finally, si-PIG-C decreased MSLN and PIG-C levels. To conclude, our investigation unravels a new axis in TNBC, where miR-182 can manipulate MSLN and PIG-C. Meanwhile, MALAT-1 is the culprit lncRNA in this novel axis, possibly a sponge for miR-182. Altogether, this sheds light on new targets for BC immune-therapy.