Obesity-associated epigenetic alterations and the obesity-breast cancer axis.

Both breast cancer and obesity can regulate epigenetic changes or be regulated by epigenetic changes. Due to the well-established link between obesity and an increased risk of developing breast cancer, understanding how obesity-mediated epigenetic changes affect breast cancer pathogenesis is critical. Researchers have described how obesity and breast cancer modulate the epigenome individually and synergistically. In this review, the epigenetic alterations that occur in obesity, including DNA methylation, histone, and chromatin modification, accelerated epigenetic age, carcinogenesis, metastasis, and tumor microenvironment modulation, are discussed. Delineating the relationship between obesity and epigenetic regulation is vital to furthering our understanding of breast cancer pathogenesis.

Targeting Mcl-1 by a small molecule NSC260594 for triple-negative breast cancer therapy.

Triple-negative breast cancers (TNBCs) are aggressive forms of breast cancer and tend to grow and spread more quickly than most other types of breast cancer. TNBCs can neither be targeted by hormonal therapies nor the antibody trastuzumab that targets the HER2 protein. There are urgent unmet medical needs to develop targeted drugs for TNBCs. We identified a small molecule NSC260594 from the NCI diversity set IV compound library. NSC260594 exhibited dramatic cytotoxicity in multiple TNBCs in a dose-and time-dependent manner. NSC260594 inhibited the Myeloid cell leukemia-1 (Mcl-1) expression through downregulation of Wnt signaling proteins. Consistent with this, NSC260594 treatment increased apoptosis, which was confirmed by using an Annexin-V/PI assay. Interestingly, NSC260594 treatment reduced the cancer stem cell (CSC) population in TNBCs. To make NSC260594 more clinically relevant, we treated NSC260594 with TNBC cell derived xenograft (CDX) mouse model, and with patient-derived xenograft (PDX) organoids. NSC260594 significantly suppressed MDA-MB-231 tumor growth in vivo, and furthermore, the combination treatment of NSC260594 and everolimus acted synergistically to decrease growth of TNBC PDX organoids. Together, we found that NSC260594 might serve as a lead compound for triple-negative breast cancer therapy through targeting Mcl-1.

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

LKB1 signaling and patient survival outcomes in hepatocellular carcinoma.

The liver is a major organ that is involved in essential biological functions such as digestion, nutrient storage, and detoxification. Furthermore, it is one of the most metabolically active organs with active roles in regulating carbohydrate, protein, and lipid metabolism. Hepatocellular carcinoma is a cancer of the liver that is associated in settings of chronic inflammation such as viral hepatitis, repeated toxin exposure, and fatty liver disease. Furthermore, liver cancer is the most common cause of death associated with cirrhosis and is the 3rd leading cause of global cancer deaths. LKB1 signaling has been demonstrated to play a role in regulating cellular metabolism under normal and nutrient deficient conditions. Furthermore, LKB1 signaling has been found to be involved in many cancers with most reports identifying LKB1 to have a tumor suppressive role. In this review, we use the KMPlotter database to correlate RNA levels of LKB1 signaling genes and hepatocellular carcinoma patient survival outcomes with the hopes of identifying potential biomarkers clinical usage. Based on our results STRADß, CAB39L, AMPKα, MARK2, SIK1, SIK2, BRSK1, BRSK2, and SNRK expression has a statistically significant impact on patient survival.

A combination of novel NSC small molecule inhibitor along with doxorubicin inhibits proliferation of triple-negative breast cancer through metabolic reprogramming.

Treatment of patients with triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and the highly invasive and proliferative nature of TNBC cells. Current treatments against TNBC have shown little promise due to high recurrence rate in patients. Consequently, there is a pressing need for novel and efficacious therapies against TNBC. Here, we report the discovery of a novel small molecule inhibitor (NSC33353) with potent anti-tumor activity against TNBC cells. The anti-proliferative effects of this small molecule inhibitor were determined using 2D and 3D cell proliferation assays. We found that NSC33353 significantly reduces the proliferation of TNBC cells in these assays. Using proteomics, next generation sequencing (NGS), and gene enrichment analysis, we investigated global regulatory pathways affected by this compound in TNBC cells. Proteomics data indicate a significant metabolic reprograming affecting both glycolytic enzymes and energy generation through oxidative phosphorylation. Subsequently, using metabolic (Seahorse) and enzymatic assays, we validated our proteomics and NGS analysis findings. Finally, we showed the inhibitory and anti-tumor effects of this small molecule in vitro and confirmed its inhibitory activity in vivo. Doxorubicin is one of the most effective agents in the treatment of TNBC and resistance to this drug has been a major problem. We show that the combination of NSC33353 and doxorubicin suppresses the growth of TNBC cells synergistically, suggesting that NSC33353 enhances TNBC sensitivity to doxorubicin. In summary, our data indicate that the small molecule inhibitor, NSC33353, exhibits anti-tumor activity in TNBC cells, and works in a synergistic fashion with a well-known chemotherapeutic agent.

Expression of Novel Kinase MAP3K19 in Various Cancers and Survival Correlations.

Mitogen Activated Protein (MAP) kinases are a category of serine/threonine kinases that have been demonstrated to regulate intracellular events including stress responses, developmental processes, and cancer progression Although many MAP kinases have been extensively studied in various disease processes, MAP3K19 is an understudied kinase whose activities have been linked to lung disease and fibroblast development. In this manuscript, we use bioinformatics databases starBase, GEPIA, and KMPlotter, to establish baseline expressions of MAP3K19 in different tissue types and its correlation with patient survival in different cancers.

Ceritinib is a novel triple negative breast cancer therapeutic agent.

BACKGROUND: Triple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12-55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment. METHODS: Ceritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR- and AR low mouse xenograft and patient derived xenograft models. RESULTS: We screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone. CONCLUSIONS: To improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.

NR4A Family Genes: A Review of Comprehensive Prognostic and Gene Expression Profile Analysis in Breast Cancer.

This report analyzes nuclear receptor (NR) subfamily 4A's potential role in treating those diagnosed with breast cancer. Here we reviewed the current literature on NR4 family members. We also examined the relative gene expression of the NR4A receptor subfamily in the basal, HER2 (human epidermal growth factor receptor 2) positive, luminal A, and luminal B subtypes using data from tumor samples in The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). These data showed a positive link between NR4A1-NR4A3 expression and increased overall survival and relapse-free survival in breast cancer patients. In addition, we observed that high expression of NR4A1, NR4A2, and NR4A3 led to better survival. Furthermore, NR4A family genes seem to play an essential regulatory role in glycolysis and oxidative phosphorylation in breast cancer. The novel prognostic role of the NR4A1-NR4A3 receptors implicates these receptors as important mediators controlling breast cancer metabolic reprograming and its progression. The review establishes a strong clinical basis for the investigation of the cellular, molecular, and physiological roles of NR4A genes in breast cancer.

Hippo signaling pathway: A comprehensive gene expression profile analysis in breast cancer.

Breast cancer (BC) is the most frequently diagnosed malignancy in women and a major public health concern. The Hippo pathway is an evolutionarily conserved signaling pathway that serves as a key regulator for a wide variety of biological processes. Hippo signaling has been shown to have both oncogenic and tumor-suppressive functions in various cancers. Core components of the Hippo pathway consist of various kinases and downstream effectors such as YAP/TAZ. In the current report, differential expression of Hippo pathway elements as well as the correlation of Hippo pathway mRNAs with various clinicopathologic characteristics, including molecular subtypes, receptor status, and methylation status, has been investigated in BC using METABRIC and TCGA datasets. In this review, we note deregulation of several Hippo signaling elements in BC patients. Moreover, we see examples of negative correlations between methylation of Hippo genes and mRNA expression. The expression of Hippo genes significantly varies between different receptor subgroups. Because of the clear associations between mRNA expression and methylation status, DNA methylation may be one of the mechanisms that regulate the Hippo pathway in BC cells. Differential expression of Hippo genes among various BC molecular subtypes suggests that Hippo signaling may function differently in different subtypes of BC. Our data also highlights an interesting link between Hippo components' transcription and ER negativity in BC. In conclusion, substantial deregulation of Hippo signaling components suggests an important role of these genes in breast cancer.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model.

Nischarin (Nisch) is a cytosolic scaffolding protein that harbors tumor-suppressor-like characteristics. Previous studies have shown that Nisch functions as a scaffolding protein and regulates multiple biological activities. In the current study, we prepared a complete Nisch knockout model, for the first time, by deletion of exons 5 and 6. This knockout model was confirmed by Qrt-PCR and Western blotting with products from mouse embryonic fibroblast (MEF) cells. Embryos and adult mice of knockouts are significantly smaller than their wild-type counterparts. Deletion of Nisch enhanced cell migration, as demonstrated by wound type and transwell migration assays. Since the animals were small in size, we investigated Nisch's effect on metabolism by conducting several assays using the Seahorse analyzer system. These data indicate that Nisch null cells have lower oxygen consumption rates, lower ATP production, and lower levels of proton leak. We examined the expression of 15 genes involved in lipid and fat metabolism, as well as cell growth, and noted a significant increase in expression for many genes in Nischarin null animals. In summary, our results show that Nischarin plays an important physiological role in metabolic homeostasis.

Role of Nischarin in the pathology of diseases: a special emphasis on breast cancer.

Nischarin has been demonstrated to have tumor suppressor functions. In this review, we comprehensively discuss up to date information about Nischarin. In addition, this paper aims to report the prognostic value, clinical relevance, and biological significance of the Nischarin gene (NISCH) in breast cancer (BCa) patients using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) datasets. We evaluated NISCH gene expression and its correlation to patient survival, baseline expression, and expression variation based on age groups, tumor stage, tumor size, tumor grade, and lymph node status in different subtypes of BCa. Since NISCH has been extensively reported to inhibit EMT and cancer cell migration, we also checked for the correlation between NISCH and EMT genes in addition to the correlation between NISCH and cell migration genes. Our results indicate that NISCH is a tumor suppressor that plays a critical role in BCa initiation, progression, and tumor development. We find that there is a higher level of NISCH expression in normal breast tissues compared to breast cancer tissues. Also, aggressive subtypes of breast cancers, such as the triple negative/basal category, have decreased levels of NISCH as the disease progresses. Finally, we report that NISCH is inversely correlated with many EMT and cancer cell migration genes in BCa. Interestingly, we identified a significant negative correlation between NISCH expression and its methylation in breast cancer patients. Overall, the goal of this report is to establish a strong clinical basis for further investigation into the cellular, molecular, and physiological roles of NISCH in BCa. Ultimately, NISCH gene expression might be clinically harnessed as a biomarker or predictor of invasiveness and metastasis in BCa.

Hippo pathway: Regulation, deregulation and potential therapeutic targets in cancer.

Hippo pathway is a master regulator of development, cell proliferation, stem cell function, tissue regeneration, homeostasis, and organ size control. Hippo pathway relays signals from different extracellular and intracellular events to regulate cell behavior and functions. Hippo pathway is conserved from Protista to eukaryotes. Deregulation of the Hippo pathway is associated with numerous cancers. Alteration of the Hippo pathway results in cell invasion, migration, disease progression, and therapy resistance in cancers. However, the function of the various components of the mammalian Hippo pathway is yet to be elucidated in detail especially concerning tumor biology. In the present review, we focused on the Hippo pathway in different model organisms, its regulation and deregulation, and possible therapeutic targets for cancer treatment.

Understanding the role of integrins in breast cancer invasion, metastasis, angiogenesis, and drug resistance.

Integrins are cell adhesion receptors, which are typically transmembrane glycoproteins that connect to the extracellular matrix (ECM). The function of integrins regulated by biochemical events within the cells. Understanding the mechanisms of cell growth by integrins is important in elucidating their effects on tumor progression. One of the major events in integrin signaling is integrin binding to extracellular ligands. Another event is distant signaling that gathers chemical signals from outside of the cell and transmit the signals upon cell adhesion to the inside of the cell. In normal breast tissue, integrins function as checkpoints to monitor effects on cell proliferation, while in cancer tissue these functions altered. The combination of tumor microenvironment and its associated components determines the cell fate. Hypoxia can increase the expression of several integrins. The exosomal integrins promote the growth of metastatic cells. Expression of certain integrins is associated with increased metastasis and decreased prognosis in cancers. In addition, integrin-binding proteins promote invasion and metastasis in breast cancer. Targeting specific integrins and integrin-binding proteins may provide new therapeutic approaches for breast cancer therapies. This review will examine the current knowledge of integrins' role in breast cancer.

Repurposing existing drugs for the treatment of COVID-19/SARS-CoV-2 infection: A review describing drug mechanisms of action.

The outbreak of a novel coronavirus (SARS-CoV-2) has caused a major public health concern across the globe. SARS-CoV-2 is the seventh coronavirus that is known to cause human disease. As of September 2020, SARS-CoV-2 has been reported in 213 countries and more than 31 million cases have been confirmed, with an estimated mortality rate of ∼3%. Unfortunately, a drug or vaccine is yet to be discovered to treat COVID-19. Thus, repurposing of existing cancer drugs will be a novel approach in treating COVID-19 patients. These drugs target viral replication cycle, viral entry and translocation to the nucleus. Some can enhance innate antiviral immune response as well. Hence this review focuses on comprehensive list of 22 drugs that work against COVID-19 infection. These drugs include fingolimod, colchicine, N4-hydroxycytidine, remdesivir, methylprednisone, oseltamivir, icatibant, perphanizine, viracept, emetine, homoharringtonine, aloxistatin, ribavirin, valrubicin, famotidine, almitrine, amprenavir, hesperidin, biorobin, cromolyn sodium, and antibodies- tocilzumab and sarilumab. Also, we provide a list of 31 drugs that are predicted to function against SARS-CoV-2 infection. In summary, we provide succinct overview of various therapeutic modalities. Among these 53 drugs, based on various clinical trials and literature, remdesivir, nelfinavir, methylpredinosolone, colchicine, famotidine and emetine may be used for COVID-19. SIGNIFICANCE: It is of utmost important priority to develop novel therapies for COVID-19. Since the effect of SARS-CoV-2 is so severe, slowing the spread of diseases will help the health care system, especially the number of visits to Intensive Care Unit (ICU) of any country. Several clinical trials are in works around the globe. Moreover, NCI developed a recent and robust response to COVID-19 pandemic. One of the NCI's goals is to screen cancer related drugs for identification of new therapies for COVID-19. https://www.cancer.gov/news-events/cancer-currents-blog/2020/covid-19-cancer-nci-response?cid=eb_govdel.

Combination treatment of bicalutamide and curcumin has a strong therapeutic effect on androgen receptor-positive triple-negative breast cancers.

Triple-negative breast cancers account for approximately 15-20% of breast cancer patients. Due to lack of expression of estrogen receptor, PR and human epidermal growth factor receptor 2 in triple-negative breast cancers, there are no targeted therapies available for these cancers. Therefore, a major research priority is to find potential therapeutic targets. Androgen receptor is present in 80-90% of all breast cancers, including 55% of estrogen receptor-α-negative cancers and 12%-35% of triple-negative breast cancers. Androgen receptor stimulates growth and survival in triple-negative breast cancer cells. Treatment with bicalutamide, an androgen receptor antagonist, has a good benefit for AR triple-negative breast cancer patients. AR triple-negative breast cancer cells were treated with curcumin or bicalutamide alone or in combination of both together. Cell growth, apoptosis and Wnt signaling pathways were examined. We found that curcumin dramatically suppressed Wnt signaling pathway in AR triple-negative breast cancer cells. Curcumin treatment inhibited androgen receptor protein expression in AR triple-negative breast cancer cells. Combination treatment of curcumin and bicalutamide has a robust increase in apoptosis. Furthermore, the combination treatment suppressed the growth of AR triple-negative breast cancer cells more effectively than with the single drug alone. Our data indicate that androgen receptor inhibition is a potential therapeutic approach for AR triple-negative breast cancers. In summary, our study for the first time shows that the combination treatment of curcumin and bicalutamide is effective in AR triple-negative breast cancer cells.

Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment.

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

Long noncoding RNAs and exosomal lncRNAs: classification, and mechanisms in breast cancer metastasis and drug resistance.

Breast cancer is the most common cancer, and the second cause of cancer-related deaths (after lung cancer) among women. Developing tumor metastasis and invasion is the most important cause of death in breast cancer patients. Several key factors participate in breast cancer metastasis including long noncoding RNAs (lncRNAs). lncRNAs are a category of cellular RNAs that are longer than 200 nucleotides in length. Accumulating evidence suggests that lncRNAs have the potential to be promising diagnostic, prognostic biomarkers and therapeutic targets in breast cancer. Understanding the role of lncRNAs and their mechanisms of functions might help to further discovery of breast cancer biological characteristics. In this review, we discuss physiological functions, epigenetic regulation, transcriptional regulation of lncRNAs, and their important role in tumor progression and metastasis. Some lncRNAs function as oncogenes and some function as tumor suppressors. Interestingly, recent reports depict that hypomethylation of promoters of lncRNAs play a pivotal role in cancer progression, suggesting the importance of epigenetic regulation. Furthermore, we discuss the role of lncRNAs in exosomes and their function in drug resistance, and therapeutic importance of exosomal lncRNAs in cancer biology. In summary, lncRNAs have a great potential to consider them as novel prognostic biomarkers as well as new therapeutic targets in breast cancer.

Exosomes: composition, biogenesis, and mechanisms in cancer metastasis and drug resistance.

Tumor-derived exosomes (TDEs) participate in formation and progression of different cancer processes, including tumor microenvironment (TME) remodeling, angiogenesis, invasion, metastasis and drug-resistance. Exosomes initiate or suppress various signaling pathways in the recipient cells via transmitting heterogeneous cargoes. In this review we discuss exosome biogenesis, exosome mediated metastasis and chemoresistance. Furthermore, tumor derived exosomes role in tumor microenvironment remodeling, and angiogenesis is reviewed. Also, exosome induction of epithelial mesenchymal transition (EMT) is highlighted. More importantly, we discuss extensively how exosomes regulate drug resistance in several cancers. Thus, understanding exosome biogenesis, their contents and the molecular mechanisms and signaling pathways that are responsible for metastasis and drug-resistance mediated by TDEs may help to devise novel therapeutic approaches for cancer progression particularly to overcome therapy-resistance and preventing metastasis as major factors of cancer mortality.

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth.

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. SIGNIFICANCE: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099.