Galectin-3-binding protein inhibits extracellular heparan 6-O-endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Avocado-derived extracellular vesicles loaded with ginkgetin and berberine prevent inflammation and macrophage foam cell formation.

Atherosclerosis, a chronic inflammatory disease of aorta, remains the major cause of morbidity and mortality among cardiovascular disease patients. Macrophage foam cell formation and inflammation are critically involved in early stages of atherosclerosis, hence chemopreventive targeting of foam cell formation by nutraceuticals may be a promising approach to curbing the progression of atherosclerosis. However, many nutraceuticals including berberine and ginkgetin have low stability, tissue/cell penetration and bioavailability resulting in inadequate chemotherapeutic effects of these nutraceuticals. We have used avocado-derived extracellular vesicles (EV) isolated from avocado (EVAvo ) as a novel carrier of nutraceuticals, in a strategy to alleviate the build-up of macrophage foam cells and expression of inflammatory genes. Our key findings are: (i) Avocado is a natural source of plant-derived EVs as shown by the results from transmission electron microscopy, dynamic light scattering and NanoBrook Omni analysis and atomic force microscopy; (ii) EVAvo are taken up by macrophages, a critical cell type in atherosclerosis; (iii) EVAvo can be loaded with high amounts of ginkgetin and berberine; (iv) ginkgetin plus berberine-loaded EVAvo (EVAvo(B+G) ) suppress activation of NFκB and NLRP3, and inhibit expression of pro-inflammatory and atherogenic genes, specifically Cd36, Tnfα, Il1ß and Il6; (v) EVAvo(B+G) attenuate oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation and (vi) EVAvo(B+G) inhibit oxLDL uptake but not its cell surface binding during foam cell formation. Overall, our results suggest that using EVAvo as a natural carrier of nutraceuticals may improve strategies to curb the progression of atherosclerosis by limiting inflammation and pro-atherogenic responses.

Heparan-6-O-Endosulfatase 2 Promotes Invasiveness of Head and Neck Squamous Carcinoma Cell Lines in Co-Cultures with Cancer-Associated Fibroblasts.

Local invasiveness of head and neck squamous cell carcinoma (HNSCC) is a complex phenomenon supported by interaction of the cancer cells with the tumor microenvironment (TME). We and others have shown that cancer-associated fibroblasts (CAFs) are a component of the TME that can promote local invasion in HNSCC and other cancers. Here we report that the secretory enzyme heparan-6-O-endosulfatase 2 (Sulf-2) directly affects the CAF-supported invasion of the HNSCC cell lines SCC35 and Cal33 into Matrigel. The Sulf-2 knockout (KO) cells differ from their wild type counterparts in their spheroid growth and formation, and the Sulf-2-KO leads to decreased invasion in a spheroid co-culture model with the CAF. Next, we investigated whether a fucosylated chondroitin sulfate isolated from the sea cucumber Holothuria floridana (HfFucCS) affects the activity of the Sulf-2 enzyme. Our results show that HfFucCS not only efficiently inhibits the Sulf-2 enzymatic activity but, like the Sulf-2 knockout, inhibits Matrigel invasion of SCC35 and Cal33 cells co-cultured with primary HNSCC CAF. These findings suggest that the heparan-6-O-endosulfatases regulate local invasion and could be therapeutically targeted with the inhibitory activity of a marine glycosaminoglycan.

Mechanosensing and Mechanosignal Transduction in Atherosclerosis.

PURPOSE OF REVIEW: This review aims to summarize the latest findings on mechanosensing in atherosclerosis, elucidating the molecular mechanisms, cellular players, and potential therapeutic targets. RECENT FINDINGS: Atherosclerosis, a chronic inflammatory disease characterized by the buildup of lipid-laden plaque within arterial walls, is a major contributor to cardiovascular disease-related mortality and morbidity. Interestingly, atherosclerosis predominantly occurs in arterial areas with curves and branches. In these regions, endothelial cells encounter irregular blood flow with distinctive low-intensity fluctuating shear stress. On the other hand, straight sections of arteries, subjected to a consistent flow and related high-intensity, one-way shear stress, are relatively safeguarded against atherosclerosis due to shear-dependent, disease-preventing endothelial cell reactions. In recent years, researchers have been investigating the role of mechanosensing in the development and progression of atherosclerosis. At the core of mechanosensing is the ability of various cells to sense and respond to biomechanical forces in their environment. In the context of atherosclerosis, endothelial cells, smooth muscle cells, and immune cells are subjected to various mechanical or physical stimuli, including shear stress, cyclic strain, and matrix stiffness. These mechanical cues play a crucial role in regulating cellular behavior and contribute to the pathophysiology of atherosclerosis. Accumulating evidence suggests that various mechanical or physical cues play a critical role in the development and promotion of atherosclerosis.

Galectin-3-binding protein inhibits extracellular heparan 6-O-endosulfatse Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the secretome of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP has functional relevance, and may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

PDE4 inhibitor eliminates breast cancer stem cells via noncanonical activation of mTOR.

Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3',5' monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.

Role of mechanosensitive channels/receptors in atherosclerosis.

Mechanical forces are critical physical cues that can affect numerous cellular processes regulating the development, tissue maintenance, and functionality of cells. The contribution of mechanical forces is especially crucial in the vascular system where it is required for embryogenesis and for maintenance of physiological function in vascular cells including aortic endothelial cells, resident macrophages, and smooth muscle cells. Emerging evidence has also identified a role of these mechanical cues in pathological conditions of the vascular system such as atherosclerosis and associated diseases like hypertension. Of the different mechanotransducers, mechanosensitive ion channels/receptors are gaining prominence due to their involvement in numerous physiological and pathological conditions. However, only a handful of potential mechanosensory ion channels/receptors have been shown to be involved in atherosclerosis, and their precise role in disease development and progression remains poorly understood. Here, we provide a comprehensive account of recent studies investigating the role of mechanosensitive ion channels/receptors in atherosclerosis. We discuss the different groups of mechanosensitive proteins and their specific roles in inflammation, endothelial dysfunction, macrophage foam cell formation, and lesion development, which are crucial for the development and progression of atherosclerosis. Results of the studies discussed here will help in developing an understanding of the current state of mechanobiology in vascular diseases, specifically in atherosclerosis, which may be important for the development of innovative and targeted therapeutics for this disease.

PIWI-interacting RNA (piRNA): a narrative review of its biogenesis, function, and emerging role in lung cancer.

Cancer remains elusive in many aspects, especially in its causes and control. After protein profiling, genetic screening, and mutation studies, scientists now have turned their attention to epigenetic modulation. This new arena has brought to light the world of noncoding RNA (ncRNA). Although very complicated and often confusing, ncRNA domains are now among the most attractive molecular markers for epigenetic control of cancer. Long ncRNA and microRNA (miRNA) have been studied best among the noncoding genome and huge data have accumulated regarding their inhibitory and promoting effects in cancer. Another sector of ncRNAs is the world of PIWI-interacting RNAs (piRNAs). Initially discovered with the asymmetric division of germline stem cells in the Drosophila ovary, piRNAs have a unique capability to associate with mammalian proteins analogous to P-element induced wimpy testis (PIWI) in Drosophila and are capable of silencing transposons. After a brief introduction to its discovery timelines, the present narrative review covers the biogenesis, function, and role of piRNAs in lung cancer. The effects on lung cancer are highlighted under sections of cell proliferation, stemness maintenance, metastasis, and overall survival, and the review concludes with a discussion of recent discoveries of another class of small ncRNAs, the piRNA-like RNAs (piR-Ls).

Lactoferrin-tethered betulinic acid nanoparticles promote rapid delivery and cell death in triple negative breast and laryngeal cancer cells.

Cancer management presents multifarious problems. Triple negative breast cancer (TNBC) is associated with inaccurate prognosis and limited chemotherapeutic options. Betulinic acid (BA) prevents angiogenesis and causes apoptosis of TNBC cells. NIH recommends BA for rapid access in cancer chemotherapy because of its cell-specific toxicity. BA however faces major challenges in therapeutic practices due to its limited solubility and cellular entree. We report lactoferrin (Lf) attached BA nanoparticles (Lf-BAnp) for rapid delivery in triple negative breast (MDA-MB-231) and laryngeal (HEp-2) cancer cell types. Lf association was confirmed by SDS-PAGE and FT-IR analysis. Average hydrodynamic size of Lf-BAnp was 147.7 ± 6.20 nm with ζ potential of -28.51 ± 3.52 mV. BA entrapment efficiency was 75.38 ± 2.70% and the release mechanism followed non-fickian pattern. Impact of Lf-BAnp on cell cycle and cytotoxicity of triple negative breast cancer and its metastatic site laryngeal cancer cell lines were analyzed. Lf-BAnp demonstrated strong anti-proliferative and cytotoxic effects, along with increased sub-G1 population and reduced number of cells in G1 and G2/M phases of the cell cycle, confirming reduced cell proliferation and significant cell death. Speedy intracellular entry of Lf-BAnp occurred within 30 min. Lf-BAnp design was explored for the first time as safer chemotherapeutic arsenals against complex TNBC conditions.

Enhancing Chemosensitivity of Breast Cancer Stem Cells by Downregulating SOX2 and ABCG2 Using Wedelolactone-encapsulated Nanoparticles.

A major caveat in the treatment of breast cancer is disease recurrence after therapeutic regime at both local and distal sites. Tumor relapse is attributed to the persistence of chemoresistant cancer stem cells (CSC), which need to be obliterated along with conventional chemotherapy. Wedelolactone, a naturally occurring coumestan, demonstrates anticancer effects in different cancer cells, although with several limitations, and is mostly ineffective against CSCs. To enhance its biological activity in cancer cells and additionally target the CSCs, wedelolactone-encapsulated PLGA nanoparticles (nWdl) were formulated. Initial results indicated that nanoformulation of wedelolactone not only increased its uptake in breast cancer cells and the CSC population, it enhanced drug retention and sustained release within the cells. Enhanced drug retention was achieved by downregulation of SOX2 and ABCG2, both of which contribute to drug resistance of the CSCs. In addition, nWdl prevented epithelial-to-mesenchymal transition, suppressed cell migration and invasion, and reduced the percentage of breast cancer stem cells (BCSC) in MDA-MB-231 cells. When administered in combination with paclitaxel, which is known to be ineffective against BCSCs, nWdl sensitized the cells to the effects of paclitaxel and reduced the percentage of ALDH+ BCSCs and mammospheres. Furthermore, nWdl suppressed growth of solid tumors in mice and also reduced CD44+/CD24-/low population. Taken together, our data imply that nWdl decreased metastatic potential of BCSCs, enhanced chemosensitivity through coordinated regulation of pluripotent and efflux genes, and thereby provides an insight into effective drug delivery specifically for obliterating BCSCs.

Stemness and chemoresistance are imparted to the OC cells through TGFß1 driven EMT.

Ovarian cancer (OC) is the fourth most common gynecological malignancy due to its highly aggressive, recurrent, and drug-resistant nature. The last two features are rendered by the presence of cancer stem cells (CSCs). Factors like TGFß1 and their downstream signaling pathways are upregulated in most cancers and are known to induce EMT and stemness, but the exact mechanisms underlying the process remain unelucidated. In our study, TGFß1 induced enhanced stem-like properties like high expression of the pluripotent markers SOX2, OCT4a, and NANOG, along with CD44, and CD117 in the OC cells. In addition, increased activity of the aldehyde dehydrogenase enzyme, formation of compact spheroids, and a quiescent phenotype were observed. In deciphering the mechanism behind it, our data propose ZEB1 transcription factor to play a substantial role in inducing the EMT-mediated stemness and chemoresistance. Further, in our study, we elucidated the significant contribution of both Smad and non-Smad pathways like ERK, JNK, and P38 MAPK pathways in the induction of stem-like characteristics. The novelty of the study also resides with the fact in the expression of different lineage-specific markers, like CD31, CD45, and CD117 along with CD44 in the TGFß1-induced epithelial ovarian cancer spheroids. This suggests a tendency of the spheroidal cells towards differentiating into heterogenic populations, which is a distinctive feature of a stem cell. Taken together, the present study provides an insight to the molecular cues involved in the acquisition of stemness and chemoresistance along with tumor heterogeneity in TGFß1-induced OC cells.

Modulation of SOX2 expression delineates an end-point for paclitaxel-effectiveness in breast cancer stem cells.

Tumor relapse in triple negative breast cancer patients has been implicated to chemoresistant cancer stem cells (CSCs), which under favorable conditions culminate in tumor re-formation and metastasis. Hence, eradication of CSCs during systemic chemotherapy is imperative. CSCs were sorted using immuno-phenotyping and aldefluor assay. Gene expression profiling of normal breast stem cells and breast CSCs from chemo-treated patients were carried out. Silencing SOX2 was achieved by siRNA method. Mammosphere culture and wound healing assays were carried out to assess efficacy of CSCs. Microarray analysis revealed elevated expression of SOX2, ABCG2 and TWIST1, unraveling an intertwined pluripotency-chemoresistance-EMT axis. Although paclitaxel treatment led to temporary arrest of cell migration, invasiveness resumed after drug removal. The 'twist in the tale' was a consistently elevated expression of TWIST1, substantiating that TWIST1 can also promote stemness and chemoresistance in tumors; hence, its eradication was imperative. Silencing SOX2 increased chemo-sensitivity and diminished sphere formation, and led to TWIST1 down regulation. This study eventually established that SOX2 silencing of CSCs along with paclitaxel treatment reduced SOX2-ABCG2-TWIST1 expression, disrupted sphere forming capacity and also reduced invasiveness by retaining epithelial-like properties of the cells, thereby suggesting a more comprehensive therapy for TNBC patients in future.

Deregulated E2F5/p38/SMAD3 Circuitry Reinforces the Pro-Tumorigenic Switch of TGFß Signaling in Prostate Cancer.

Transforming growth factor-ß signaling exerts divergent effects on normal and cancer cells, although mechanism underlying this differential behavior remains unclear. In this study, expression of 94 genes pertaining to the TGF-ß signaling pathway was compared between tumor and benign tissue samples from the human prostate gland to identify major discriminators driving prostate carcinogenesis. E2F5 was identified as one of the most deregulated genes in prostate cancer tissues, predominantly in samples with Gleason-score 6. Expression of other deregulated components of TGF-ß signaling was examined by qRT-PCR, Western blot, and immune-staining. Function of E2F5 and p38 in prostate cancer was investigated using siRNA-treatment of PC3 cell-line followed by analyses of associated components and cell cycle. Observations revealed that E2F5 overexpression was accompanied by significantly higher phosphorylation of SMAD3 at Ser-208 in the linker region (pSMAD3L) and p38 in tumor tissue. A striking difference in SMAD3 phosphorylation, marked by preponderance of pSMAD3L and pSMAD3C (Ser-423 and 425) in tumor and benign tissues, respectively was noted. Co-localization of E2F5 with pSMAD3L in the nuclei of tumor and PC3 cells indicated a functional interface between the proteins. Downregulation of E2F5 and p38 in PC3 cells resulted in marked reduction of phosphorylation of SMAD3 and perturbation of cell cycle with an arrest of cells in G1 . Our findings unearthed that E2F5/p38 axis played a cardinal role in uncontrolled cellular proliferation in prostate cancer through pSMAD3L activation. It also underscores a strong potential for E2F5 to be incorporated as a tool in early detection of prostate cancer. J. Cell. Physiol. 231: 2482-2492, 2016. © 2016 Wiley Periodicals, Inc.