Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2–4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7–10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)—a secreted glycoprotein aberrantly abundant in different cancers—as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.     

Fluorophore‐Labeled Cyclooxygenase‐2 Inhibitors for the Imaging of Cyclooxygenase‐2 Overexpression in Cancer: Synthesis and Biological Studies

A group of cyclooxygenase‐2 (COX‐2)‐specific fluorescent cancer biomarkers were synthesized by linking the anti‐inflammatory drugs ibuprofen, (S)‐naproxen, and celecoxib to the 7‐nitrobenzofurazan (NBD) fluorophore. In vitro COX‐1/COX‐2 inhibition studies indicated that all of these fluorescent conjugates are COX‐2 inhibitors (IC₅₀ range: 0.19–23.0 μM ) with an appreciable COX‐2 selectivity index (SI≥4.3–444). In this study the celecoxib–NBD conjugate N‐(2‐((7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)amino)ethyl)‐4‐(5‐(p‐tolyl)‐3‐(trifluoromethyl)‐1H‐pyrazol‐1‐yl)benzenesulfonamide ( 14 ), which displayed the highest COX‐2 inhibitory potency and selectivity (COX‐2 IC₅₀=0.19 μM ; SI=443.6), was identified as an impending COX‐2‐specific biomarker for the fluorescence imaging of cancer using a COX‐2‐expressing human colon cancer cell line (HCA‐7).     

The Journey of DDR1 and DDR2 Kinase Inhibitors as Rising Stars in the Fight Against Cancer

Discoidin domain receptor (DDR) is a collagen-activated receptor tyrosine kinase that plays critical roles in regulating essential cellular processes such as morphogenesis, differentiation, proliferation, adhesion, migration, invasion, and matrix remodeling. As a result, DDR dysregulation has been attributed to a variety of human cancer disorders, for instance, non-small-cell lung carcinoma (NSCLC), ovarian cancer, glioblastoma, and breast cancer, in addition to some inflammatory and neurodegenerative disorders. Since the target identification in the early 1990s to date, a lot of efforts have been devoted to the development of DDR inhibitors. From a medicinal chemistry perspective, we attempted to reveal the progress in the development of the most promising DDR1 and DDR2 small molecule inhibitors covering their design approaches, structure-activity relationship (SAR), biological activity, and selectivity.     

Selective and Brain‐Permeable Polo‐like Kinase‐2 (Plk‐2) Inhibitors That Reduce α‐Synuclein Phosphorylation in Rat Brain

Polo‐like kinase‐2 (Plk‐2) has been implicated as the dominant kinase involved in the phosphorylation of α‐synuclein in Lewy bodies, which are one of the hallmarks of Parkinson’s disease neuropathology. Potent, selective, brain‐penetrant inhibitors of Plk‐2 were obtained from a structure‐guided drug discovery approach driven by the first reported Plk‐2–inhibitor complexes. The best of these compounds showed excellent isoform and kinome‐wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk‐2 inhibition in vivo. One such compound significantly decreased phosphorylation of α‐synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson’s disease.     

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.     

Identification of Activated Protein Kinase Cα (PKCα) in the Urine of Orthotopic Bladder Cancer Xenograft Model as a Potential Biomarker for the Diagnosis of Bladder Cancer

Bladder cancer has a high recurrence rate; therefore, frequent and effective monitoring is essential for disease management. Cystoscopy is considered the gold standard for the diagnosis and continuous monitoring of bladder cancer. However, cystoscopy is invasive and relatively expensive. Thus, there is a need for non-invasive, relatively inexpensive urinary biomarker-based diagnoses of bladder cancer. This study aimed to investigate the presence of activated protein kinase Cα (PKCα) in urine samples and the possibility of PKCα as a urinary biomarker for bladder cancer diagnosis. Activated PKCα was found to be present at higher levels in bladder cancer tissues than in normal bladder tissues. Furthermore, high levels of activated PKCα were observed in urine samples collected from orthotopic xenograft mice carrying human bladder cancer cells compared to urine samples from normal mice. These results suggest that activated PKCα can be used as a urinary biomarker to diagnose bladder cancer. To the best of our knowledge, this is the first report describing the presence of activated PKCα in the urine of orthotopic xenograft mice.     

Synthesis and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 1,3,4,5‐Tetrasubstituted 1H‐Pyrrol‐2(5H)‐one Scaffold

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito‐borne pathogen that causes a large number of human infections each year. There are currently no vaccines or antiviral therapies available for human use against WNV. Therefore, efforts to develop new chemotherapeutics against this virus are highly desired. In this study, a WNV NS2B–NS3 protease inhibitor with a 1,3,4,5‐tetrasubstituted 1H‐pyrrol‐2(5H)‐one scaffold was identified by screening a small library of nonpeptidic compounds. Optimization of this initial hit by the synthesis and screening of a focused library of compounds with this scaffold led to the identification of a novel uncompetitive inhibitor ((?)‐ 1a16 , IC₅₀=2.2±0.7 μM ) of the WNV NS2B–NS3 protease. Molecular docking of the chiral compound onto the WNV protease indicates that the R enantiomer of 1a16 interferes with the productive interactions between the NS2B cofactor and the NS3 protease domain and is thus the preferred isomer for inhibition of the WNV NS2B–NS3 protease.     

Regulation of the Soluble Amyloid Precursor Protein α (sAPPα) Levels by Acetylcholinesterase and Brain-Derived Neurotrophic Factor in Lung Cancer Cell Media

In comparing two human lung cancer cells, we previously found lower levels of acetylcholinesterase (AChE) and intact amyloid-β40/42 (Aβ), and higher levels of mature brain-derived neurotrophic factor (mBDNF) in the media of H1299 cells as compared to A549 cell media. In this study, we hypothesized that the levels of soluble amyloid precursor protein α (sAPPα) are regulated by AChE and mBDNF in A549 and H1299 cell media. The levels of sAPPα were higher in the media of H1299 cells. Knockdown of AChE led to increased sAPPα and mBDNF levels and correlated with decreased levels of intact Aβ40/42 in A549 cell media. AChE and mBDNF had opposite effects on the levels of Aβ and sAPPα and were found to operate through a mechanism involving α-secretase activity. Treatment with AChE decreased sAPPα levels and simultaneously increased the levels of intact Aβ40/42 suggesting a role of the protein in shifting APP processing away from the non-amyloidogenic pathway and toward the amyloidogenic pathway, whereas treatment with mBDNF led to opposite effects on those levels. We also show that the levels of sAPPα are regulated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3 Kinase (PI3K), but not by protein kinase A (PKA).