We previously reported that Ganoderma lucidum extract (GLE) demonstrate significant anti-cancer activity against triple negative inflammatory breast cancer models. Herein, we aimed to elucidate the bioactive compounds of GLE responsible for this anti-cancer activity. We performed NMR, X-ray crystallography and analog derivatization as well as anti-cancer activity studies to elucidate and test the compounds. We report the structures of the seven most abundant GLE compounds and their selective efficacy against triple negative (TNBC) and inflammatory breast cancers (IBC) and other human cancer cell types (solid and blood malignancies) to illustrate their potential as anti-cancer agents. Three of the seven compounds (ergosterol, 5,6-dehydroergosterol and ergosterol peroxide) exhibited significant in vitro anti-cancer activities, while we report for the first time the structure elucidation of 5,6-dehydroergosterol from Ganoderma lucidum . We also show for the first time in TNBC/IBC cells that ergosterol peroxide (EP) displays anti-proliferative effects through G1 phase cell cycle arrest, apoptosis induction via caspase 3/7 activation, and PARP cleavage. EP decreased migratory and invasive effects of cancer cells while inhibiting the expression of total AKT1, AKT2, BCL-XL, Cyclin D1 and c-Myc in the tested IBC cells. Our investigation also indicates that these compounds induce reactive oxygen species, compromising cell fate. Furthermore, we generated a superior derivative, ergosterol peroxide sulfonamide, with improved potency in IBC cells and ample therapeutic index (TI > 10) compared to normal cells. The combined studies indicate that EP from Ganoderma lucidum extract is a promising molecular scaffold for further exploration as an anti-cancer agent.

The aggressive nature of triple negative breast cancer (TNBC) may be explained in part by the presence of breast cancer stem cells (BCSCs), a subpopulation of cells, which are involved in tumor initiation, progression, metastasis, recurrence, and therapy resistance. The signal transducer and activator of transcription 3 (STAT3) pathway participates in the development and progression of BCSCs, but its role in TNBC remains unclear. Here, we report that Ganoderma lucidum extract (GLE), a medicinal mushroom with anticancer activity, acts on BCSCs in vitro and in TNBC pre-clinical animal tumor models by downregulating the STAT3 pathway. We show that GLE significantly reduces TNBC cell viability, and down-regulates total and phosphorylated STAT3 expression. This is consistent with the reduction of OCT4, NANOG and SOX2 expression, reduction in the BCSC population by loss of the ALDH1 and CD44+/CD24– population, the deformation of mammospheres, and the strong reduction in animal tumor volume and tumor weight. Analysis of the BCSC compartment in tumors revealed that GLE decreases the STAT3 pathway and the expression of OCT4, NANOG, and SOX2 in BCSCs. These findings demonstrate that the anti-cancer activity of GLE targets BCSCs of TNBC through the downregulation of the STAT3 pathway.

The first total synthesis of a C5-Curcumin-2-Hexadecynoic Acid (C5-Curc-2-HDA, 6) conjugate was successfully performed. Through a three-step synthetic route, conjugate 6 was obtained in 13 % overall yield and tested for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. Our results revealed that 6 was active against eight MRSA strains at MICs that range between 31.3 and 62.5 μg/mL. It was found that the presence of 2-hexadecynoic acid (2-HDA, 4) in conjugate 6 increased 4-8-fold its antibacterial activity against MRSA strains supporting our hypothesis that the chemical connection of 4 to C5-Curcumin (2) increases the antibacterial activity of 2 against Gram-positive bacteria. Combinational index (CIn) values that range between 1.6 and 2.3 were obtained when eight MRSA strains were treated with an equimolar mixture of 2 and 4. These results demonstrated that an antagonistic effect is taking place. Finally, it was investigated whether conjugate 6 can affect the replication process of S. aureus, since this compound inhibited the supercoiling activity of the S. aureus DNA gyrase at minimum inhibitory concentrations (MIC) of 250 μg/mL (IC50 = 100.2 ± 13.9 μg/mL). Moreover, it was observed that the presence of 4 in conjugate 6 improves the anti-topoisomerase activity of 2 towards S. aureus DNA gyrase, which is in agreement with results obtained from antibacterial susceptibility tests involving MRSA strains.

Breast cancer (BC) is the second leading cause of cancer death among women worldwide. The main cause of BC morbidity and mortality is the invasiveness capacity of cancer cells that may lead to metastasis. Here, we aimed to investigate the therapeutic efficacy of Ganoderma lucidum extract (GLE)—a medicinal mushroom with anticancer properties—on BC motility via the Rac/Lamellipodin pathway. GLE treatment effects were tested on MDA-MB-231 breast cancer cells. The effects were tested on cell viability, migration and invasion. Pulldowns, immunoblotting, and immunofluorescence were used to measure Rac activity and the expression of proteins involved in cell migration and in lamellipodia formation, respectively. As a result, GLE suppressed BC cell viability, migration, and invasion capacity. GLE impaired Rac activity, as well as downregulated Lamellipodin, ENA/VASP, p-FAK (Tyr925), Cdc42, and c-Myc expression. Lamellipodia formation was significantly reduced by GLE. In conclusion, we demonstrate that GLE reduces Rac activity and downregulates signaling molecules involved in lamellipodia formation. These novel findings serve as basis for further studies to elucidate the potential of GLE as a therapeutic agent regulating the Rac/Lamellipodin pathway in BC metastasis.

The first synthesis of C5-Curcumin-Fatty Acid (C5-Curc-FA) conjugates was successfully performed. Through a two-step synthetic route, 10 analogs were synthesized for a structure-activity relationship (SAR) study. It was found that C5-Curc-FA conjugates containing either decanoic acid or palmitic acid moieties were cytotoxic against colorectal adenocarcinoma cell (CCL-229) at IC50s ranging from 22.5 to 56.1 µg/mL, being 5c the most active C5-Curc-FA conjugate. Our results strongly suggests that a decanoic acid moiety at the meta position in C5-Curc-FA conjugates is important for their anticancer activity effect. Possible mechanisms for the anticancer activity of C5-Curc-FA conjugates were also investigated including apoptosis induction, mitochondrial damage and caspases activation. It was shown that 5c inhibited the luminescence activity of NFκB, a key signaling molecule involved in cell apoptosis and cell proliferation, at IC50 = 18.2 µg/mL. In addition, it was demonstrated that 5c displayed significant apoptotic effect at GI50 = 46.0 µg/mL in colorectal adenocarcinoma cell line (ATCC CCL-222), which can be explained by the significant mitochondrial membrane permeabilization and caspases 3 and 7 activation effect of 5c. Finally, it was investigated that C5-Curc-FA conjugates can affect the replication process of cancer cells, since compounds 5c, 5e, and 6c inhibited the relaxing activity of the human DNA topoisomerase I at minimum inhibitory concentrations (MICs) that range from 50 to 500 µg/mL. Our results strongly support the hypothesis that the inhibition of both NFkB and DNA topoisomerase I by C5-Curc-FA conjugates is associated with their anticancer activity.

Accumulation of amyloid in breast cancer is a well-known phenomenon, but only immunoglobulin light-chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Ab), is present in an aggregated form in animal and human highgrade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Ab peptides and in what form, we used a nude mouse model inoculated with triple-negative inflammatory breast cancer cell (SUM-149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Ab identified the clear presence of Ab peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Ab peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse-generated Ab40 in tumors. We conclude that Ab in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Ab accumulation in tumors and whether this phenomenon could represent the specific signature of highgrade cancers.Inflammatory breast cancer (IBC) is one of the most aggressive types of this disease; it is highly metastatic and usually fatal (as it is commonly diagnosed at T4, according to the tumor-nodes-metastasis classification of cancers (TNM) classification). Various IBC carcinoma cell subtypes exist, with the triple-negative subtype the most frequently diagnosed. These tumors lack receptors for estrogen, progesterone, or human epidermal growth factor receptor 2 (proto-oncogene) (HER2/neu), and thus, IBC is often treated with chemotherapy, radiation, or Abbreviations AD, Alzheimer's disease; AL, light-chain amyloidosis; APP, amyloid precursor protein; Ab, amyloid beta; DAPI-4 0 , 6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; HER2/neu, human epidermal growth factor receptor 2 (proto-oncogene); IBC, inflammatory breast cancer; SUM-149-IBC cell line; MALT, mucosa-associated lymphoid tissue; MAPK, mitogen-activated protein kinases; PDGF, plateletderived growth factor; SCID, severe combined immunodeficient (mice strain); TNM, tumor-nodes-metastasis classification of cancers; TTR, transthyretin.

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer where patients have a 43% increased risk of death compared to women with non‐IBC. The lethality of IBC originates from its nature of invading the vascular and lymphatic systems, absence of a typical tumor mass, and the generation of tumor emboli. The arising challenges in IBC include the accurate and early diagnosis and the development of effective targeted therapies. A previous study investigated potential biomarkers involved in tumor spheroid integrity by characterizing IBC’s cell surface proteome. Plasma membrane proteins were identified via “stable isotope labeling with amino acids in cell culture” (SILAC) with subsequent mass spectrometry (MS)‐based quantitative proteomics analysis using the IBC cell line SUM‐149 vs. non‐ cancerous mammary epithelial cells, MCF10A. SILAC results showed overexpression of Metadherin (MTDH) in SUM‐149 cells (>2.0 fold change). Subsequently, we validated the overexpression of MTDH in SUM‐149 IBC cells, IBC tissues and in the emboli. MTDH is a cell adhesion molecule overexpressed in many cancer types, including breast cancer. MTDH promotes cancer progression by modulating various signaling pathways including NF‐kB, which is related to cell survival, proliferation, invasion and metastasis. Therefore, the aim of this study is to assess the functional role of MTDH in IBC progression. To elucidate the mechanism of action of MTDH in colony formation and tumor spheroid we silenced MTDH (CRISPR or shRNA) SUM‐149 and SUM‐190 IBC cells. We assessed the expression of MTDH by immunoblotting and immunofluorescence where we observed reduced MDTH levels. The number of colonies formed in SUM‐149 MTDH silenced cells was ~40% lower when compared to non‐silenced cells. Tumor spheroids of SUM‐149 and SUM‐190 MTDH silenced cells were smaller than non‐silenced cells. Aditionally, MTDH silencing results in a decrease in the phosphorylation of the p65 subunit of NF‐kB while no changes are observed in total protein abundance in SUM‐149 cells. We can conclude from our preliminary results that MTDH serves as a potential IBC target for IBC progression. Support or Funding Information This work was supported by NIH NIGMS #GM111171 (MMM), SGRP 2017‐00143 (MMM and GOS), NIMHD #MD007583 (MMM), GM103475 (UPR MMM), Title‐V‐PPOHA #P031M105050, Title‐V‐Cooperative #P031S130068 from the U.S. Dept. of Education and Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health #P20GM103475 (GOS).

Breast cancer (BC) is the leading cause of cancer among women in the United States and Puerto Rico. Triple‐negative breast cancer (TNBC) is characterized by the absence of estrogen and progesterone receptors, and lack of HER2 overexpression. Approximately, 15% of all women with TNBC typically display shorter overall survival and an early peak of distant recurrences at 3y after diagnosis. Treatments for TNBC are limited; currently, there are no approved targeted therapies besides non‐targeted chemotherapy. The mortality of TNBC could result from the lack of effective treatments to decrease the migration of cancer cells to other tissues. Hence, it its highly important to understand these migration processes and to find effective therapies. Therefore, we aimed to test the anti‐migration effects of the medicinal mushroom Ganoderma lucidumextract (GLE) on TNBC cell motility and on the regulation of proteins involved in this process. First, MDA‐MB‐231 cells were treated with various GLE concentrations and evaluated for cell viability and a washout assay. To study the effect of GLE on cell migration, cells were treated with vehicle, 0.25 or 0.96mg/mL (IC50) of GLE for 24h, followed by wound healing and invasion assays. The activity of Rac was tested by a pulldown activity assay and immunoblotting. Furthermore, we tested the expression of proteins important in the dynamics of cell motility Cdc42, Lamellipodin (Lpd), FAK, WAVE and Rho. The localization and expression of Lpd was visualized by immunocytochemistry. Results showed that GLE significantly decreased the cell viability on TNBC, with no cell recovery after a 72h period of treatment withdrawal. GLE significantly decreased MDA‐MB‐231 cell migration and invasion, while reducing the activity of Rac. The expression of Lpd, FAK, WAVE, c‐myc, and Cdc 42 were also modulated upon GLE treatment. GLE decreased Lpd localization in ruffled edges of cancer cells, as well as changes in nuclear and cell morphology. In conclusion, our results suggest the potential of GLE as a natural anti‐TNBC migration agent.Support or Funding InformationThis work was supported by grants from the National Institutes of Health GM111171 (MMM), MD007583 (MMM), GM103475 INBRE‐Supplies for Graduate Students (GOS), from the Puerto Rico Science, Technology and Research Trust (PRSTRT) SGRP 2017‐043, PRSTRT Hurricane Relief Funds, and from the U.S. Dept of Education Title‐V‐PPOHA P031M105050 and Title‐V‐Cooperative P031S130068. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Puerto Rico Science, Technology and Research Trust or the U.S. Department of Education.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.