Pentagalloyl Glucose (PGG) Exhibits Anti-Cancer Activity against Aggressive Prostate Cancer by Modulating the ROR1 Mediated AKT-GSK3ß Pathway.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-ß-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.

Identification and validation of a small molecule targeting ROR1 for the treatment of triple negative breast cancer.

Introduction: Breast cancer is the most common cancer in women, with roughly 10-15% of new cases classified as triple-negative breast cancer (TNBC). Traditional chemotherapies are often toxic to normal cells. Therefore, it is important to discover new anticancer compounds that target TNBC while causing minimal damage to normal cells. Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is an oncofetal protein overexpressed in numerous human malignancies, including TNBC. This study investigated potential small molecules targeting ROR1. Methodology: Using AutoDock Vina and Glide, we screened 70,000 chemicals for our investigation. We obtained 10 representative compounds via consensus voting, deleting structural alerts, and clustering. After manual assessment, compounds 2 and 4 were chosen for MD simulation and cell viability experiment. Compound 4 showed promising results in the viability assay, which led us to move further with the apoptosis assay and immunoblotting. Results: Compound 4 (CID1261330) had docking scores of -6.635 and -10.8. It fits into the pocket and shows interactions with GLU64, ASP174, and PHE93. Its RMSD fluctuates around 0.20 nm and forms two stable H-bonds indicating compound 4 stability. It inhibits cell proliferation in MDA-MB-231, HCC1937, and HCC1395 cell lines, with IC50 values of approximately 2 µM to 10 µM, respectively. Compound 4 did not kill non-malignant epithelial breast cells MCF-10A (IC50 > 27 µM). These results were confirmed by the significant number of apoptotic cells in MDA-MB-231 cells (47.6%) but not in MCF-10A cells (7.3%). Immunoblot analysis provided additional support in the same direction. Discussion: These findings collectively suggest that compound 4 has the potential to effectively eliminate TNBC cells while causing minimal harm to normal breast cells. The promising outcomes of this study lay the groundwork for further testing of compound 4 in other malignancies characterized by ROR1 upregulation, serving as a proof-of-concept for its broader applicability.

Receptor tyrosine kinase-like orphan receptor 1 inhibitor strictinin exhibits anti-cancer properties against highly aggressive androgen-independent prostate cancer.

Aim: It is important to identify anti-cancer compounds that can inhibit specific molecular targets to eradicate androgen-receptor negative (ARneg), androgen-independent (AI) prostate cancer, which is an aggressive form of prostate cancer with limited treatment options. The goal of this study was to selectively target prostate cancer cells that have high levels of oncogenic protein Receptor tyrosine kinase-like orphan receptor 1 (ROR1) by using strictinin, a small molecule ROR1 inhibitor. Methods: The methods performed in this study include western blots, methyl thiazolyl tetrazolium (MTT) proliferation assays, phosphatidylserine apoptosis assays, apoptosis flow cytometry (Annexin V, caspase 3/7), migration scratch assays, Boyden chamber invasion assays, and cell cycle flow cytometry. Results: Strictinin was most lethal against PC3 [half-maximal drug inhibitory concentration (IC50) of 277.2 µmol/L], an ARneg-AI cell type that expresses the highest levels of ROR1. Strictinin inhibited ROR1 expression, downstream phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3beta (GSK3ß) pro-survival signaling, and epithelial-to-mesenchymal transition markers in PC3 cells. Additionally, strictinin decreased PC3 cell migration and invasion, while increasing S-phase cell cycle arrest. In ARneg-AI DU145 cells, strictinin inhibited ROR1 expression and modulated downstream AKT-GSK3ß signaling. Furthermore, strictinin exhibited anti-migratory, anti-invasive, but minimal pro-apoptotic effects in DU145 cells likely due to DU145 having less ROR1 expression in comparison to PC3 cells. Throughout the study, strictinin minimally impacted the phenotype of normal prostatic epithelial cells RWPE-1 (IC50 of 658.5 µmol/L). Strictinin was further identified as synergistic with docetaxel [combination index (CI) = 0.311] and the combination therapy was found to reduce the IC50 of strictinin to 38.71 µmol/L in PC3 cells. Conclusions: ROR1 is an emerging molecular target that can be utilized for treating prostate cancer. The data from this study establishes strictinin as a potential therapeutic agent that targets ARneg-AI prostate cancer with elevated ROR1 expression to reduce the migration, invasion, cell cycle progression, and survival of prostate cancer.

Structure-activity relationship of 2-aminodibenzothiophene pharmacophore and the discovery of aminobenzothiophenes as potent inhibitors of Mycobacterium smegmatis.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide and its current treatments have been complicated with the emergence of multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains. Therefore, the discovery of new antitubercular agents is in need to overcome this problem. In our efforts to discover novel candidates for the treatment of tuberculosis, we describe in this work in vitro activityagainstM. smegmatis for a series of aminated benzo-fused heterocycles, particularly, dibenzothiophene to explore the structure-activity relationship of 2-aminodibenzothiophene 3aa. From these studies, three compounds 5-aminobenzothiophene 3ia, 6-aminobenzothiophene 3ma (MIC: 0.78 µg/mL) and 5-aminobenzofuran 3ja (MIC: 1.56 µg/mL) were identified as potent inhibitors of M. smegmatis with low cytotoxicity. These results suggested the significance of these compounds 3ia, 3ja and 3ma for the future development of candidate agents to treat tuberculosis.

Protein Tyrosine Phosphatases: Mechanisms in Cancer.

Protein tyrosine kinases, especially receptor tyrosine kinases, have dominated the cancer therapeutics sphere as proteins that can be inhibited to selectively target cancer. However, protein tyrosine phosphatases (PTPs) are also an emerging target. Though historically known as negative regulators of the oncogenic tyrosine kinases, PTPs are now known to be both tumor-suppressive and oncogenic. This review will highlight key protein tyrosine phosphatases that have been thoroughly investigated in various cancers. Furthermore, the different mechanisms underlying pro-cancerous and anti-cancerous PTPs will also be explored.

Emerging Immunotherapies against Novel Molecular Targets in Breast Cancer.

Immunotherapy is a highly emerging form of breast cancer therapy that enables clinicians to target cancers with specific receptor expression profiles. Two popular immunotherapeutic approaches involve chimeric antigen receptor-T cells (CAR-T) and bispecific antibodies (BsAb). Briefly mentioned in this review as well is the mRNA vaccine technology recently popularized by the COVID-19 vaccine. These forms of immunotherapy can highly select for the tumor target of interest to generate specific tumor lysis. Along with improvements in CAR-T, bispecific antibody engineering, and therapeutic administration, much research has been done on novel molecular targets that can especially be useful for triple-negative breast cancer (TNBC) immunotherapy. Combining emerging immunotherapeutics with tumor marker discovery sets the stage for highly targeted immunotherapy to be the future of cancer treatments. This review highlights the principles of CAR-T and BsAb therapy, improvements in CAR and BsAb engineering, and recently identified human breast cancer markers in the context of in vitro or in vivo CAR-T or BsAb treatment.

Regulation of cancer stem cells in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC) is the most lethal subtype of breast cancer. Despite the successes of emerging targeted therapies, relapse, recurrence, and therapy failure rates in TNBC significantly outpace other subtypes of breast cancer. Mounting evidence suggests accumulation of therapy resistant Cancer Stem Cell (CSC) populations within TNBCs contributes to poor clinical outcomes. These CSCs are enriched in TNBC compared to non-TNBC breast cancers. The mechanisms underlying CSC accumulation have been well-characterized and discussed in other reviews. In this review, we focus on TNBC-specific mechanisms that allow the expansion and activity of self-renewing CSCs. We highlight cellular signaling pathways and transcription factors, specifically enriched in TNBC over non-TNBC breast cancer, contributing to stemness. We also analyze publicly available single-cell RNA-seq data from basal breast cancer tumors to highlight the potential of emerging bioinformatic approaches in identifying novel drivers of stemness in TNBC and other cancers.

Retinoic acid induces differentiation in neuroblastoma via ROR1 by modulating retinoic acid response elements.

Neuroblastoma is the most common inheritable, solid neoplasm in children found under the age of 7 and accounts for approximately 7% of childhood cancers. A common treatment that has been prescribed for over a decade is retinoid therapy [using all­trans retinoic acid (RA)]. Treatment with this differentiating agent has been revealed to progress the cells from their stem­cell state to a mature neuronal state gaining classical neuronal characteristics, including the suppression of proliferation. However, the molecular mechanism underlying the action of RA treatment remains to be elucidated. In the present study, a novel mechanism of RA­induced differentiation via regulation of receptor tyrosine kinase­like orphan receptor 1 (ROR1) is reported. ROR1 is overexpressed in neuroblastoma but significantly downregulated in mature differentiated neurons. Hence, it was hypothesized that RA may modulate ROR1 leading to differentiation and termination of cancerous properties. Immunoblotting revealed that following RA treatment, ROR1 levels initially increased then sharply decreased by 96 h. This was paired with synaptophysin, a mature neuron marker, sharply increasing concurrently, providing evidence of differentiation by 96 h. Investigation of the ROR1 pathway confirmed ROR1­dependent downstream activation of the PI3K/AKT signaling axis, a growth pathway previously demonstrated to promote differentiation. Chromatin immunoprecipitation revealed an increase in RAR binding to the promoters of ROR1 and its endogenous ligand, Wnt5a. This research provided compelling evidence that RA is able to modulate the expression of ROR1 and Wnt5a to promote differentiation through the expression of synaptophysin. This data combined with the overarching data from the scientific community regarding proliferation and other proliferative factors in early­stage neurons provides a more in­depth model of the process of differentiation in neurons.

ROR1 regulates chemoresistance in Breast Cancer via modulation of drug efflux pump ABCB1.

Chemoresistance is one of the leading causes of mortality in breast cancer (BC). Understanding the molecules regulating chemoresistance is critical in order to combat chemoresistant BC. Drug efflux pump ABCB1 is overexpressed in chemoresistant neoplasms where it effluxes various chemotherapeutic agents from cells. Because it is expressed in normal and cancerous cells alike, attempts at targeting ABCB1 directly have failed due to low specificity and disruption of normal tissue. A proposed method to inhibit ABCB1 is to target its cancer-specific, upstream regulators, mitigating damage to normal tissue. Few such cancer-specific upstream regulators have been described. Here we characterize ROR1 as an upstream regulator of ABCB1. ROR1 is highly expressed during development but not expressed in normal adult tissue. It is however highly expressed in several cancers. ROR1 is overexpressed in chemoresistant BC where it correlates with poor therapy response and tumor recurrence. Our data suggests, ROR1 inhibition sensitizes BC cells to chemo drugs. We also show ROR1 regulates ABCB1 stability and transcription via MAPK/ERK and p53. Validating our overall findings, inhibition of ROR1 directly correlated with decreased efflux of chemo-drugs from cells. Overall, our results highlight ROR1's potential as a therapeutic target for multidrug resistant malignancies.

Strictinin, a novel ROR1-inhibitor, represses triple negative breast cancer survival and migration via modulation of PI3K/AKT/GSK3ß activity.

Triple Negative Breast Cancer (TNBC), the most aggressive subtype of breast cancer, is characterized by the absence of hormone receptors usually targeted by hormone therapies like Tamoxifen. Because therapy success and survival rates for TNBC lag far behind other breast cancer subtypes, there is significant interest in developing novel anti-TNBC agents that can target TNBC specifically, with minimal effects on non-malignant tissue. To this aim, our study describes the anti-TNBC effect of strictinin, an ellagitanin previously isolated from Myrothamnus flabellifolius. Using various in silico and molecular techniques, we characterized the mechanism of action of strictinin in TNBC. Our results suggest strictinin interacts strongly with Receptor Tyrosine Kinase Orphan like 1 (ROR1). ROR1 is an oncofetal receptor highly expressed during development but not in normal adult tissue. It is highly expressed in several human malignancies however, owing to its numerous pro-tumor functions. Via its interaction and inhibition of ROR1, strictinin reduced AKT phosphorylation on ser-473, inhibiting downstream phosphorylation and inhibition of GSK3ß. The reduction in AKT phosphorylation also correlated with decreased cell survival and activation of the caspase-mediated intrinsic apoptotic cascade. Strictinin treatment also repressed cell migration and invasion in a beta-catenin independent manner, presumably via the reactivated GSK3ß's repressing effect on microtubule polymerization and focal adhesion turnover. This could be of potential therapeutic interest considering heightened interest in ROR1 and other receptor tyrosine kinases as targets for development of anti-cancer agents. Further studies are needed to validate these findings in other ROR1-expressing malignancies but also in more systemic models of TNBC. Our findings do however underline the potential of strictinin and other ROR1-targeting agents as therapeutic tools to reduce TNBC proliferation, survival and motility.

Leaf biomechanical properties in Arabidopsis thaliana polysaccharide mutants affect drought survival.

Individual sugars are the building blocks of cell wall polysaccharides, which in turn comprise a plant׳s overall architectural structure. But which sugars play the most prominent role in maintaining a plant׳s mechanical stability during large cellular deformations induced by drought? We investigated the individual contributions of several genes that are involved in the synthesis of monosaccharides which are important for cell wall structure. We then measured drought tolerance and mechanical integrity during simulated drought in Arabidopsis thaliana. To assess mechanical properties, we designed a small-scale tensile tester for measuring failure strain, ultimate tensile stress, work to failure, toughness, and elastic modulus of 6-week-old leaves in both hydrated and drought-simulated states. Col-0 mutants used in this study include those deficient in lignin, cellulose, components of hemicellulose such as xylose and fucose, the pectic components arabinose and rhamnose, as well as mutants with enhanced arabinose and total pectin content. We found that drought tolerance is correlated to the mechanical and architectural stability of leaves as they experience dehydration. Of the mutants, S096418 with mutations for reduced xylose and galactose was the least drought tolerant, while the arabinose-altered CS8578 mutants were the least affected by water loss. There were also notable correlations between drought tolerance and mechanical properties in the diminished rhamnose mutant, CS8575 and the dehydrogenase-disrupted S120106. Our findings suggest that components of hemicellulose and pectins affect leaf biomechanical properties and may play an important role in the ability of this model system to survive drought.

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize.

BACKGROUND: Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. RESULTS: In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. CONCLUSIONS: CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.