Steven McClellan

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.

miR-125b Functions as a Key Mediator for Snail-induced Stem Cell Propagation and Chemoresistance

Background: Snail plays an important role in chemoresistance, but the mechanism is still unclear. Results: Up-regulation of microRNA-125b through Wnt signaling by snail enriches cancer stem cells and increases chemoresistance. Conclusion: MicroRNA-125b is a key mediator for Snail-induced stem cell propagation and chemoresistance. Significance: We reveal a novel mechanism for Snail-induced stem cell maintenance and chemoresistance. Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/β-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

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.

Panepoxydone Targets NF-kB and FOXM1 to Inhibit Proliferation, Induce Apoptosis and Reverse Epithelial to Mesenchymal Transition in Breast Cancer

Background Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines. Methods We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. Results Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. Conclusion Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.

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.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1a

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

ABSTRACT Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

mRNA detection in living cells: A next generation cancer stem cell identification technique

Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technology that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers associated with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examined by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.

ErbB2-intronic microRNA-4728: a novel tumor suppressor and antagonist of oncogenic MAPK signaling.

Although the role of the ErbB2/HER2 oncogene in cancers has been extensively studied, how ErbB2 is regulated remains poorly understood. A novel microRNA, mir-4728, was recently found within an intron of the ErbB2 gene. However, the function and clinical relevance of this intronic miRNA are completely unknown. Here, we demonstrate that mir-4728 is a negative regulator of MAPK signaling through directly targeting the ERK upstream kinase MST4 and exerts numerous tumor-suppressive properties in vitro and in animal models. Importantly, our patient sample study shows that mir-4728 was under-expressed in breast tumors compared with normal tissue, and loss of mir-4728 correlated with worse overall patient survival. These results strongly suggest that mir-4728 is a tumor-suppressive miRNA that controls MAPK signaling through targeting MST4, revealing mir-4728’s significance as a potential prognostic factor and target for therapeutic intervention in cancer. Moreover, this study represents a conceptual advance by providing strong evidence that a tumor-suppressive miRNA can antagonize the canonical signaling of its host oncogene.