Tumor Suppressor Protein PTEN Research Articles

Apigenin promotes cell death in NCI-H23 cells by upregulation of PTEN: potential involvement of the binding of apigenin with WWP2 protein

The tumour suppressor protein PTEN is often down-regulated in non-small cell lung cancer. A major protein promoting the lowering of the PTEN protein is WWP2. Polyphenols have been shown to promote the expression of tumour suppressor genes like PTEN. We carry out the study to check for the ability of apigenin to bind with the WWP2 protein using in-silico investigation comprising docking and simulation. We checked for the cytotoxic effect of apigenin upon the non-small cell lung cancer cell line NCI-H23. We checked the PTEN expression status at the gene and protein levels. The expression levels of the apoptotic regulators BCL2, BAX and CASPASE3 genes along with the activity levels of the caspase-3 protein were checked. The ultrastructure of the cells was analysed. Our Autodock analysis showed that apigenin bound favourably with the WWP2 protein. Molecular dynamics simulation revealed that apigenin increased the parameters of RMSD, Rg and SASA when bound with the WWP2 protein. The protein-ligand complex had hydrogen bonding and majorly van der Wal’s interactions. PCA analysis revealed greater fluctuations in the apigenin-bound state of the protein. The mutant form of the WWP2 revealed similar results in the presence of apigenin. Apigenin showed efficacy against the NCI-H23 cell line and promoted PTEN protein levels, lowered BCL2 gene expression and up-regulated BAX and CASPASE3 gene expression. Increased caspase-3 activity and ultra-structural analysis revealed the occurrence of apoptosis. Thus the binding of apigenin with WWP2 could promote PTEN protein levels and lead to apoptotic activity in NCI-H23 cells. Communicated by Ramaswamy H. Sarma

Localisation of phosphoinositides in the grass endophyte Epichloë festucae and genetic and functional analysis of key components of their biosynthetic pathway in E. festucae symbiosis and Fusarium oxysporum pathogenesis

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals.

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKTSer473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.

Persistent activation of the mammalian target of rapamycin signalling pathway in cutaneous squamous cell carcinomas in cats.

Cutaneous squamous cell carcinoma (CSCC) represents the most common malignant tumour of the feline skin. Emerging evidence suggests that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway may represent a potential target for pharmacological intervention in human and canine CSCC. The present study aimed to explore the expression pattern and status of activation of relevant signalling proteins of the PI3K/Akt/mTOR signalling pathway in feline CSCC. The expression of pEGFRTyr1068 , pAktSer473 , pS6Ser235/236 combined with Ki-67, and the tumour suppressor protein PTEN was evaluated by immunohistochemical analysis in 45 samples of feline CSCC, using a tissue microarray. The immunodetection using phosphospecific antibodies to detect the activated forms of signalling proteins showed that the PI3K/Akt/mTOR signalling pathway is frequently activated in feline CSCCs, and may be independent of the activation of EGFR. The results also showed that PTEN expression is not significantly altered in feline CSCCs. Our study shows that the persistent activation of the PI3K/Akt/mTOR signalling pathway represents a key event in feline CSCC, pointing to this signalling pathway being a potential therapeutic target in feline patients with CSCC.

Melanoma-specific expression of the tumor suppressor proteins p16 and PTEN is a favorable prognostic factor in established melanoma brain metastases.

PTEN and p16 frequently undergo (epi)genetic aberrations in melanoma resulting in decreased, or absent, protein levels. We investigated the prognostic significance of these tumor suppressor genes in melanoma brain metastases (MBMs). Immunohistochemical analysis was performed on archived tissue sections from craniotomies. Expression of PTEN and p16 was semiquantitatively scored (0-3 scale) in melanoma cells, glia, TILs, and endothelial cells of tumor-associated vessels and was compared among the different brain tumor cell compartments. Overall survival (OS) analysis was performed according to PTEN and p16 protein expression in melanoma cells. 58 patients (median age 56, 37 male) underwent craniotomy for MBMs before February 2014. The OS of patients with decreased, or absent, protein expression (0, 1+) of PTEN and p16 in melanoma cells was significantly shorter compared to that of patients with high (2+, 3+) expression (median OS 2.40 vs. 10.75 months and 4.1 vs. 8.1 months, respectively; Gehan-Breslow-Wilcoxon test P = 0.026 and P = 0.037, respectively). PTEN and p16 protein expression were significantly lower in TILs compared to melanoma cells (Mann-Whitney test P = 0.023 and P < 0.0001, respectively). Low/absent protein expression of PTEN/p16 is an adverse prognostic factor in MBMs. Surprisingly, expression of both PTEN and p16 proteins was significantly lower in TILs compared to melanoma cells. Proliferating (p16 absent/low) TILs within the brain with or without an active PI3K-Akt pathway (PTEN absent/low) may represent a favorable host response in MBMs. Thus, treatment of patients with MBMs with CDK4/6 or PI3K pathway inhibitors may result in an unfavorable, bystander, off-target effect on host immune response.

One Step Histological Detection and Staining of the PTEN Tumor Suppressor Protein by a Single Strand DNA

Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.

Taurine Attenuates Carcinogenicity in Ulcerative Colitis-Colorectal Cancer Mouse Model.

Taurine (2-aminoethane-sulfonic acid) is a type of amino acids and has numerous physiological and therapeutic functions, including anti-inflammation. However, there are few studies on the anticancer action of taurine. Our previous studies have demonstrated that taurine exhibits an apoptosis-inducing effect on human nasopharyngeal carcinoma cells in vitro. In this study, we have investigated whether taurine has an anticancer effect, using azoxymethane (AOM)/sulfate sodium (DSS)- induced mouse model for colon carcinogenesis. All mice, except those in control group, received a single intraperitoneal injection of AOM and DSS in the drinking water for 7 days twice, with 1-week interval. After the first DSS treatment, mice were given distilled water (model group) or taurine in the drinking water (taurine group) ad libitum. No tumor was observed in the control group. Taurine significantly suppressed AOM+DSS-induced tumor formation. Histopathological examination revealed AOM/DSS treatment induced colon cancer in all mice (8/8, 100%), and taurine significantly inhibited the progression of colon cancer (4/9, 44.4%). Taurine significantly attenuated cell proliferation in cancer tissues detected by Ki-67 staining. Taurine significantly increased the levels of an apoptosis marker cleaved caspase-9 and tumor suppressor protein PTEN. This is the first study that demonstrated that taurine significantly reduced carcinogenicity in vivo using AOM/DSS-induced colon cancer mouse model.

Safety results from phase I/II study of the PI3Kβ inhibitor GSK2636771 (G) in combination with pembrolizumab (P) in patients (pts) with PD-1 refractory metastatic melanoma (MM) and PTEN loss.

e22000 Background: Checkpoint inhibitors (CPI) have improved survival and long-term disease control in 35-40% of pts with MM. Many pts derive no clinical benefit or progress after an initial response. Our group and others have shown that loss of the tumor suppressor protein PTEN occurs in multiple cancers, up to 30% of MM pts, activates the PI3K pathway, and correlates with decreased MM response rates to CPI and decreased T cell infiltrates. In PTEN-null MM preclinical models, inhibition of the PI3Kβ-subunit with GSK2636771 (G) was superior to pan-PI3K inhibitors, increased intratumoral T cell infiltration and the activity of CPI. To test our hypothesis that PI3Kβi reverses resistance to CPI, we are conducting a Phase I/II study (NCT03131908) combining G with P in PD-1 refractory pts with PTEN loss. Methods: The primary objective of Ph I portion is to determine the Maximum-Tolerated Dose (MTD) and Recommended Phase II Dose (RP2D) of G with P in PD-1 refractory pts (including melanoma, endometrial, TNBC, and prostate cancers) with PTEN loss. Pts receive P at 200mg IV q 3 wks. G starting dose level (DL1) was 300 mg PO qd for 21 days and escalated to 400 mg PO qd (DL2) using a 3+3 design. A dose level -1 (DL-1) (200 mg PO qd) was also included in the event of unacceptable toxicities at higher doses. Ph II will accrue 35 pts at the RP2D. This study is continuously monitored for toxicity and futility. The primary objectives of Ph II are safety, tolerability, and efficacy of the combination as defined by Objective Response Rate (ORR) by RECIST 1.1. Secondary Objectives include the PKs of G and PD effects in tumor tissue as measured by pathway inhibition and T cell trafficking into tumors. Results: 13 pts have been treated, 6 at the 300mg (DL1), 5 at 400mg (DL2), and 2 at 200 mg (DL-1). One DLT (grade 3 hypocalcemia) was observed at the 300mg dose. Two DLTs were observed in the 400mg cohort, one of which was AKI requiring dialysis and the other was a Gr 3 rash. Based on this experience and additional safety data from GSK regarding renal toxicity, DL-1 was declared RP2D at 200mg. 2 pts at the RP2D have passed the DLT evaluation period without toxicities. Conclusions: The combination of G and P is being explored at the RP2D of 200 mg. Renal toxicity precluded higher doses. No objective responses have been observed although 2 pts have experienced prolonged clinical benefit including a MM pt with 27% decrease in tumor burden. Through longitudinal biopsies, we aim to better understand the role PTEN loss plays when targeted in combination with CPI. Clinical trial information: NCT03131908.

Retraction: SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT.

Retraction: SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT.

Analyzing aggregation propensities of clinically relevant PTEN mutants: a new culprit in pathogenesis of cancer and other PTENopathies

While studies on pathological protein aggregation are largely limited to neurodegenerative disease, emerging evidence suggests that other diseases are also associated with pathogenic protein aggregation. For example, tumor suppressor protein p53, and its mutant conformers, undergo protein aggregation, exacerbating the cancer phenotype. These findings raise the possibility that inactivation of tumor suppressors via protein aggregation may participate in cancer and other disease pathologies. Since tumor suppressor protein PTEN has similar functions to p53, and is mutated in multiple diseases, we examined the aggregation propensity of PTEN wild-type and 1523 clinically relevant PTEN mutants. Applying computational tools to PTEN mutation databases revealed that PTEN wild-type protein can aggregate under physiological conditions, and 274 distinct PTEN mutants had increased aggregation propensity. To understand the mechanism underlying PTEN conformer aggregation, we analyzed the physicochemical properties of these 274 PTEN mutants and defined their aggregation potential. We conclude that increased aggregation propensity of select PTEN mutants may contribute to disease phenotypes. Our studies have built the foundation for interrogating the aggregation potential of these select mutants in cancers and in PTENopathies. Elucidating the pathogenic mechanisms associated with aggregation-prone PTEN conformers will aid in developing therapies that target PTEN-aggregates in multiple diseases.Communicated by Ramaswamy H. Sarma

Expression of p53 and PTEN in human primary endometrial carcinomas: Clinicopathological and immunohistochemical analysis and study of their concomitant expression.

Endometrial carcinoma is a common malignancy of the female genital tract. Alterations in the expression levels of various oncogenes and tumor suppressor genes serve important roles in the carcinogenesis and biological behavior of endometrial carcinoma. The aim of the present study was to evaluate the combination and individual expression of p53 and phosphatase and tensin homolog (PTEN) protein in human endometrial carcinoma. In addition, the correlation of these proteins with clinicopathological parameters was also assessed. Retrospective immunohistochemical analysis of the expression of p53 and PTEN tumor suppressor proteins was conducted in 99 women with endometrial carcinoma. The overall rate of p53 and PTEN positivity was 89 and 77%, respectively, according to the sum of stain intensity and scores of immunopositive cells. The sum of p53 positivity correlated strongly with PTEN expression (ρ=0.256; P=0.044). The concomitant sum of p53 and PTEN expression was identified in 45% of patients with endometrial adenocarcinoma. Notably, the sum of the immunohistochemical expression of p53 was significantly correlated with patient age (P=0.037), histologic type (P=0.008), histologic grade (P=0.002) and fallopian and/or ovarian invasion (P=0.014). Furthermore, PTEN expression was associated with myometrial invasion (ρ=−0.377; P=0.002) and clinical stage (P=0.019). In addition, concomitant p53 and PTEN expression was correlated with patient age (P=0.008) and histologic differentiation (P=0.028). The findings indicated a correlation between the expression of p53 and PTEN in endometrial adenocarcinoma, which suggested an intrinsic association between expression levels of these tumor suppressor genes. The study also suggested that concomitant p53 and PTEN expression contributed in characterizing the tumor behavior of endometrial carcinoma. Taken together, the present study suggested the combined expression of p53 and PTEN in the development of high-grade endometrial carcinoma in older patients. In addition, the findings indicated activation of different molecular pathways in the tumor progression between low-grade and high-grade endometrial carcinomas.

Biophysical simulations and structure-based modeling of residue interaction networks in the tumor suppressor proteins reveal functional role of cancer mutation hotspots in molecular communication

In the current study, we have combined molecular simulations and energetic analysis with dynamics-based network modeling and perturbation response scanning to determine molecular signatures of mutational hotspot residues in the p53, PTEN, and SMAD4 tumor suppressor proteins. By examining structure, energetics and dynamics of these proteins, we have shown that inactivating mutations preferentially target a group of structurally stable residues that play a fundamental role in global propagation of dynamic fluctuations and mediating allosteric interaction networks. Through integration of long-range perturbation dynamics and network-based approaches, we have quantified allosteric potential of residues in the studied proteins. The results have revealed that mutational hotspot sites often correspond to high centrality mediating centers of the residue interaction networks that are responsible for coordination of global dynamic changes and allosteric signaling. Our findings have also suggested that structurally stable mutational hotpots can act as major effectors of allosteric interactions and mutations in these positions are typically associated with severe phenotype. Modeling of shortest inter-residue pathways has shown that mutational hotspot sites can also serve as key mediating bridges of allosteric communication in the p53 and PTEN protein structures. Multiple regression models have indicated that functional significance of mutational hotspots can be strongly associated with the network signatures serving as robust predictors of critical regulatory positions responsible for loss-of-function phenotype. The results of this computational investigation are compared with the experimental studies and reveal molecular signatures of mutational hotspots, providing a plausible rationale for explaining and localizing disease-causing mutations in tumor suppressor genes.

An overview on the role of dietary phenolics for the treatment of cancers.

Plant derived phenolic compounds have been shown to inhibit the initiation and progression of cancers by modulating genes regulating key processes such as: (a) oncogenic transformation of normal cells; (b) growth and development of tumors; and (c) angiogenesis and metastasis. Recent studies focusing on identifying the molecular basis of plant phenolics-induced cancer cell death have demonstrated down-regulation of: (a) oncogenic survival kinases such as PI3K and Akt; (b) cell proliferation regulators that include Erk1/2, D-type Cyclins, and Cyclin Dependent Kinases (CDKs); (c) transcription factors such as NF-kβ, NRF2 and STATs; (d) histone deacetylases HDAC1 and HDAC2; and (e) angiogenic factors VEGF, FGFR1 and MIC-1. Furthermore, while inhibiting oncogenic proteins, the phenolic compounds elevate the expression of tumor suppressor proteins p53, PTEN, p21, and p27. In addition, plant phenolic compounds and the herbal extracts rich in phenolic compounds modulate the levels of reactive oxygen species (ROS) in cells thereby regulate cell proliferation, survival and apoptosis. Furthermore, recent studies have demonstrated that phenolic compounds undergo transformation in gut microbiota thereby acquire additional properties that promote their biological activities. In vitro observations, preclinical and epidemiological studies have shown the involvement of plant phenolic acids in retarding the cancer growth. However, to date, there is no clinical trial as such testing the role of plant phenolic compounds for inhibiting tumor growth in humans. More over, several variations in response to phenolic acid rich diets-mediated treatment among individuals have also been reported, raising concerns about whether phenolic acids could be used for treating cancers. Therefore, we have made an attempt to (a) address the key structural features of phenolic acids required for exhibiting potent anti-cancer activity; (b) review the reported findings about the mechanisms of action of phenolic compounds and their transformation by gut microbiota; and (c) update the toxicological aspects and anti-tumor properties of phenolic compounds and extracts containing phenolic compounds in animals.

Enzyme-catalyzed expressed protein ligation.

Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-Cys containing peptides but the requirement of a Cys residue at the ligation junction can limit its use. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

Abstract A40: Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis

Abstract Purpose of Study: Pancreatic cancer is an overwhelming fatal disease with less than 5% of patients surviving beyond 5 years. The most prominent histopathological hallmark of pancreatic cancer is its uniquely dense stromal reaction as evidenced by recent reports that highlight the significant role of stromal fibroblasts on pancreatic tumor cell biology. We used novel mouse models to show that genetic inactivation of Smoothened (Smo) in stromal fibroblasts accelerated Kras-initiated tumorigenesis. Research Method: We used a genetically engineered mouse model of pancreatic cancer that relies on constitutive activation of the Kras oncogene in the epithelium. We simultaneously employed cre-loxP technology to conditionally delete Smo exclusively in the fibroblast compartment of the pancreas, thus disrupting the crucial hedgehog paracrine signaling loop between pancreatic tumor cells and fibroblasts. Novel Findings: We showed that deletion of Smo in stromal fibroblasts accelerated pancreatic tumorigenesis through a mechanism involving destabilization of fibroblast PTEN protein. Down-regulation of PTEN enhanced TGF-α production in stromal fibroblasts, and increased epithelial cell transformation and proliferation through epithelial growth factor receptor (EGFR). A selective SMO inhibitor also decreased PTEN in a Kras mouse model as well as in human primary pancreatic cancer associated fibroblasts. Importantly, in pancreatic ductal adenocarcinoma (PDAC) patient samples, low PTEN expression correlated with low SMO expression and with reduced overall survival. These results define a pathway that reprograms stromal fibroblasts from a tumor suppressive phenotype to a tumor promoting phenotype, thus highlighting the dual functions of stromal fibroblasts in pancreatic cancer and the molecular consequences of loss of the hedgehog pathway. Thus, a more comprehensive understanding of tumor-stroma interactions is required to assure effective implementation of targeted therapies. Conclusions and Implications: Recent pre-clinical reports suggest the pancreatic tumor microenvironment functions predominantly to inhibit tumor growth, challenging the concept of tumor stroma as a therapeutic target. Our results provide molecular insight into how the balance between the opposing activities of tumor stromal fibroblasts is maintained, and potentially identifies targets for restoring stromal tumor suppressive functions. In summary, we demonstrate that ablation of paracrine hedgehog signaling in SMA-positive fibroblasts leads to proteasome-mediated degradation of the PTEN tumor suppressor protein and subsequent activation of oncogenic pathways. Citation Format: Jason R. Pitarresi, Michael C. Ostrowski. Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A40.

A Novel Experimental Platform to Study the Temporal Evolution of Phosphoinositide Gradients in Model Membranes

A broad range of cellular functions have been linked to dynamically controlled phosphoinositide gradients, including the migration of Dictyostelium discoideum and human neutrophils. For efficient Dictyostelium migration phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) gradients are maintained through positive Ras/PI(3,4,5)P3 and negative Ras/TorC2/Akt feedback loops. Currently in the field there are several mathematical models that have been proposed to describe the dynamics of these phosphoinositide gradients. However, there are exceedingly few methods to test and expand these hypotheses using real model membranes with defined lipid and protein compositions of varying lateral heterogeneities. We have developed a novel experimental platform that takes advantage of the lamellar flow of microfluidics to study phosphoinositide gradients in supported lipid bilayers (SBL). With this platform we have successfully generated the phosphoinositide gradients in SBL. We quantified the gradients by total internal reflectance fluorescence microscopy (TIRF) and studied how these gradients are affected by external factors such as calcium. In addition, this platform can also be used to study the impact of proteins on the lipid gradient. We studied proteins such as the tumor suppressor protein PTEN that dephorphorylates PI(3,4,5)P3 to PI(4,5)P2 and how this enzyme can remodel the SBL in a gradient dependent manner. This novel platform can inform how the association of these proteins can impact such lipid gradients and begin to unravel how microorganisms maintain their complex lateral membrane organization.

Abstract B41: Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity

Abstract Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we previously reported that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins in human MDA-MB-231 TNBC cells, instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of the miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by the miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and the miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3′UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knockdown miR-17-5p reduced PDCD4 and PTEN protein expression, instead of raising them. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells also reduced PDCD4 and PTEN protein levels. To test the hypothesis that miR-17-3p passenger strand can also modulate the same mRNA targets as miR-17-5p, we performed luciferase assays using reporter constructs harboring predicted miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs. We found that the miR-17-5p mimic lowered the expression of luciferase vectors containing one predicted binding site from the PDCD4 or PTEN 3′UTR domains. However, the miR-17-3p mimic lowered the expression of luciferase vectors containing all four 3′UTR target sites of PDCD4, and two of the six predicted PTEN 3′UTR target sites. In addition, we used luciferase assays to test the hypothesis that antisense DNA-LNA against miR-17-5p mimicked passenger strand miR-17-3p. We found that anti-miR-17-5p DNA-LNA lowered the expression of luciferase reporter vectors containing two of the four predicted PDCD4 3′UTR target sites for miR-17-3p, and one of the six predicted PTEN 3′UTR target sites for miR-17-3p. From these results, we concluded that the miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs, just like the miR-17-5p guide strand. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked the miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. Supported by the Department of Biochemistry and Molecular Biology. Conflict of interest: pending PCT/US2015/015681 patent application licensed to Nikita RNA LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B41.

Intracellular delivery of the PTEN protein using cationic lipidoids for cancer therapy.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor, mutated or inactive in a large percentage of human cancers. Restoring PTEN activity in cancer cells through gene therapy has shown to inhibit cell growth and induce apoptosis, particularly in cells with a PTEN deficiency. Gene therapy, however, comes with some inherent risks such as triggering an immune response and permanent off target effects. Nanoparticle assisted protein delivery could mitigate these liabilities while maintaining therapeutic integrity. In this report, we evaluated the use of cationic lipid-like (lipidoid) materials to intracellularly deliver the PTEN protein. We synthesized a small library of cationic lipidoid materials and screened for the delivery of PTEN based on cell viability. The lipidoid material EC16-80 was selected for high efficacy and the subsequent lipidoid-protein complex was characterized using DLS, zeta potential, and TEM. Intracellular delivery of PTEN with EC16-80 to the PTEN deficient prostate cancer cell line PC-3 resulted in a significant decrease in activated AKT and induced apoptosis. Interestingly, delivery of PTEN to PTEN deficient prostate cancer cell lines PC-3 and LNCaP compared to the breast cancer cell line, MCF-7 with endogenous PTEN, resulted in significantly lower IC50 values in PC-3 and LNCaP cells indicating that the treatment is predominantly specific to PTEN-deficient cells. Altogether, these results demonstrate the first intracellular delivery of recombinant PTEN using a synthetic delivery vehicle and highlight the potential of intracellular PTEN protein delivery as a potential targeted cancer therapy.

Non-Specific Blocking of miR-17-5p Guide Strand in Triple Negative Breast Cancer Cells by Amplifying Passenger Strand Activity.

Conventional wisdom holds that only one of the two strands in a micro ribonucleic acid (miRNA) precursor duplex is selected as the active miRNA guide strand. The complementary miRNA passenger strand, however, is thought to be inactive. High levels of the oncogenic miRNA (oncomiR) guide strand called miR-17-5p is overexpressed in triple negative breast cancer (TNBC) and can inhibit ribosomal translation of tumor suppressor gene mRNAs, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, antisense knockdown of oncomiR miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human TNBC cells instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3’UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knock down miR-17-5p reduced PDCD4 and PTEN protein expression instead of raising them. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand.

Polyphyllin VII induces apoptosis in HepG2 cells through ROS-mediated mitochondrial dysfunction and MAPK pathways.

Background Paris polyphylla is an oriental folk medicine that has anticancer activities both in vivo and in vitro. Polyphyllin VII (PP7), a pennogenyl saponin from P. polyphylla has been found to exert strong anticancer activity. However, the underlying mechanisms are poorly understood. In the present study, the anticancer effect of polyphyllin VII against human liver cancer cells and the molecular mechanisms were investigated.MethodsCellular viability was measured by MTT assay. Apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential levels were evaluated using the InCell 2000 confocal microscope. The expression levels of apoptotic-related proteins were evaluated by Western blotting.ResultsPP7 strongly inhibited the cell growth and induced apoptosis and necrosis in hepatocellular carcinoma HepG2 cells. Meanwhile, PP7 up-regulated the levels of Bax/Bcl-2, cytochrome c, the cleaved forms of caspases-3, -8, -9, and poly (ADP-ribose) polymerase in a dose- and time-dependent manner, indicating that PP7 induced apoptosis in HepG2 cells through both intrinsic and extrinsic pathways. Moreover, PP7 provoked the production of intracellular ROS and the depolarization of mitochondrial membrane potential. Further analysis showed that PP7 significantly augmented the phosphorylation of JNK, ERK and p38, the major components of mitogen-activated protein kinase (MAPK) pathways, and the expressions of tumor suppressor proteins p53 and PTEN. In addition, PP7-induced apoptosis was remarkably attenuated by MAPK inhibitors and ROS inhibitor.ConclusionsThese results demonstrated that PP7 induced apoptotic cell death in HepG2 cells through both intrinsic and extrinsic pathways by promoting the generation of mitochondrial-mediated ROS and activating MAPK and PTEN/p53 pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1036-x) contains supplementary material, which is available to authorized users.