Ajay P. Singh

MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

Pancreatic cancer (PC) has the worst prognosis among all cancers due to its late diagnosis and lack of effective therapies. Therefore, identification of novel gene targets, which are differentially expressed in PC and functionally involved in malignant phenotypes, is critical to achieve early diagnosis and development of effective therapeutic strategies. We have shown previously that MUC4, an aberrantly overexpressed transmembrane mucin, promotes growth, invasion and metastasis of PC cells, thus underscoring its potential as a clinical target. Here, we report a novel microRNA (miRNA)-mediated mechanism underlying aberrant expression of MUC4 in PC. We demonstrate that the 3 untranslated region of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 downregulates endogenous MUC4 protein levels. We also show that miR-150-mediated MUC4 downregulation is associated with a concomitant decrease in human epidermal growth factor receptor 2 and its phosphorylated form, leading to reduced activation of downstream signaling. Furthermore, our findings demonstrate that miR-150 overexpression inhibits growth, clonogenicity, migration and invasion and enhances intercellular adhesion in PC cells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associated with MUC4 protein levels. Altogether, these findings establish miR-150 as a novel regulator of MUC4 and a tumor suppressor miRNA in PC.

Honokiol: A Novel Natural Agent for Cancer Prevention and Therapy

Honokiol (3,5-di-(2-propenyl)-1,1-biphenyl-2,4-diol) is a bioactive natural product derived from Magnolia spp. Recent studies have demonstrated anti-inflammatory, anti-angiogenic, anti-oxidative and anticancer properties of honokiol in vitro and in preclinical models. Honokiol targets multiple signaling pathways including nuclear factor kappa B (NF-κB), signal transducers and activator of transcription 3 (STAT3), epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (m-TOR), which have great relevance during cancer initiation and progression. Furthermore, pharmacokinetic profile of honokiol has revealed a desirable spectrum of bioavailability after intravenous administration in animal models, thus making it a suitable agent for clinical trials. In this review, we discuss recent data describing the molecular targets of honokiol and its anti-cancer activities against various malignancies in pre-clinical models. Evaluation of honokiol in clinical trials will be the next step towards its possible human applications.

Honokiol Arrests Cell Cycle, Induces Apoptosis, and Potentiates the Cytotoxic Effect of Gemcitabine in Human Pancreatic Cancer Cells

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G1 phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.

An Undesired Effect of Chemotherapy GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT, NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4

Background: CXCR4 signaling protects pancreatic cancer cells from gemcitabine toxicity. However, the effect of gemcitabine on this resistance mechanism is unclear. Results: Gemcitabine up-regulates CXCR4 expression in pancreatic cancer cells and promotes their invasiveness. Conclusion: CXCR4 signaling serves as a counterdefense mechanism against gemcitabine. Significance: These findings are significant for the formulation of effective therapeutic strategies against pancreatic cancer. Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

CXCL8 and its cognate receptors in melanoma progression and metastasis

The incidence of melanoma is rising at an alarming rate and we are still awaiting an effective treatment for this malignancy. In its early stage, melanoma can be cured by surgical removal, but once metastasis has occurred there is no effective treatment. Recent findings have suggested multiple functional implications of CXCL8 and its cognate receptors, CXCR1 and CXCR2, in melanoma pathogenesis, thus underscoring their importance as targets for cancer therapy. This review provides an update on the roles of CXCL8 and its receptors in melanoma progression and metastasis.

CXCR1 and CXCR2 silencing modulates CXCL8-dependent endothelial cell proliferation, migration and capillary-like structure formation.

CXCR1 and CXCR2 are receptors for angiogenic ELR+CXC chemokines and are differentially expressed on endothelial cells; however, their functional significance in angiogenesis remains unclear. In this study, we determined the functional significance of these receptors in modulating endothelial cell phenotype by knocking-down the expression of CXCR1 and/or CXCR2 in human microvascular endothelial cells (HMEC-1) using short-hairpin RNA (shRNA). Cell proliferation, migration, invasion and capillary-like structure (CLS) formation were analyzed. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression inhibited endothelial cell proliferation, survival, migration, invasion and CLS formation. Additionally, we examined the mechanism of CXCL8-dependent CXCR1 and/or CXCR2 mediated phenotypic changes by evaluating ERK phosphorylation and cytoskeletal rearrangement and observed inhibition of ERK phosphorylation and cytoskeletal rearrangement in HMEC-1-shCXCR1, HMEC-1-shCXCR2 and HMEC-1-shCXCR1/2 cells. Together, these data demonstrate that CXCR1 and CXCR2 expression plays a critical role in regulating multiple biological activities in human microvascular endothelial cells.

Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis.

UNLABELLEDnUltraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis.nnnFROM THE CLINICAL EDITORnExcessive exposure to the sun is known to increase the risk of skin cancer due to DNA damage. In this work, the authors tested the use of silver nanoparticles as protective agents against ultraviolet radiation. The positive results may open a door for the use of silver nanoparticle as novel agents in the future.

Myb overexpression overrides androgen depletion–induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24+/CD44+/EpCAM+) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24−/CD44−/EpCAM−) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemo-sensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

Synthesis, characterization, and evaluation of poly (D,L-lactide-co-glycolide)-based nanoformulation of miRNA-150: potential implications for pancreatic cancer therapy.

MicroRNAs are small (18–22 nucleotide long) noncoding RNAs that play important roles in biological processes through posttranscriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer. Recently, we identified miR-150 as a novel tumor suppressor microRNA in pancreatic cancer. Furthermore, expression of miR-150 was downregulated in the majority of tumor cases, suggesting that its restoration could serve as an effective approach for pancreatic cancer therapy. In the present study, we developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy in vitro. Using double emulsion solvent evaporation method, we developed a poly (D,L-lactide-co-glycolide) (PLGA)-based nanoformulation of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that these nanoparticles had high encapsulation efficiency (~78%) and exhibited sustained release profile. Treatment of pancreatic cancer cells with miR-150-NF led to efficient intracellular delivery of miR-150 mimics and caused significant downregulation of its target gene (MUC4) expression. Inhibition of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of pancreatic cancer cells with miR-150-NF suppressed their growth, clonogenicity, motility, and invasion. Together, these findings suggest that PLGA-based nanoformulation could potentially serve as a safe and effective nanovector platform for miR-150 delivery to pancreatic tumor cells.