Bilal B. Hafeez

PSMA targeted docetaxel-loaded superparamagnetic iron oxide nanoparticles for prostate cancer.

Docetaxel (Dtxl) is currently the most common therapeutic option for prostate cancer (PC). However, adverse side effects and problems associated with chemo-resistance limit its therapeutic outcome in clinical settings. A targeted nanoparticle system to improve its delivery to and activity at the tumor site could be an attractive strategy for PC therapy. Therefore, the objective of this study was to develop and determine the anti-cancer efficacy of a novel docetaxel loaded, prostate specific membrane antigen (PSMA) targeted superparamagnetic iron oxide nanoparticle (SPION) (J591-SPION-Dtxl) formulation for PC therapy. Our results showed the SPION-Dtxl formulation exhibits an optimal particle size and zeta potential, which can efficiently be internalized in PC cells. SPION-Dtxl exhibited potent anti-cancer efficacy via induction of the expression of apoptosis associated proteins, downregulation of anti-apoptotic proteins, and inhibition of chemo-resistance associated protein in PC cell lines. J591-SPION-Dtxl exhibited a profound uptake in C4-2 (PSMA(+)) cells compared to PC-3 (PSMA(-)) cells. A similar targeting potential was observed in ex-vivo studies in C4-2 tumors but not in PC-3 tumors, suggesting its tumor specific targeting. Overall, this study suggests that a PSMA antibody functionalized SPION-Dtxl formulation can be highly useful for targeted PC therapy.

miRNA nanotherapeutics for cancer.

MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression through diverse mechanisms. Increasing evidence suggests that miRNA-based therapies, either restoring or repressing miRNA expression and activity, hold great promise. However, the efficient delivery of miRNAs to target tissues is a major challenge in the transition of miRNA therapy to the clinic. Cationic polymers or viral vectors are efficient delivery agents but their systemic toxicity and immunogenicity limit their clinical usage. Efficient targeting and sustained release of miRNAs/anti-miRNAs using nanoparticles (NPs) conjugated with antibodies and/or peptides could reduce the required therapeutic dosage while minimizing systemic and cellular toxicity. Given their importance in clinical oncology, here we focus on the development of miRNA nanoformulations to achieve enhanced cellular uptake, bioavailability, and accumulation at the tumor site.

MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade, including ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility, invasion of PDAC cells—all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13–HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13–HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13–HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Cucurbitacin D exhibits potent anti-cancer activity in cervical cancer

In this study, we for the first time, investigated the potential anti-cancer effects of a novel analogue of cucurbitacin (Cucurbitacin D) against cervical cancer in vitro and in vivo. Cucurbitacin D inhibited viability and growth of cervical cancer cells (CaSki and SiHa) in a dose-dependent manner. IC50 of Cucurbitacin D was recorded at 400 nM and 250 nM in CaSki and SiHa cells, respectively. Induction of apoptosis was observed in Cucurbitacin D treated cervical cancer cells as measured by enhanced Annexin V staining and cleavage in PARP protein. Cucurbitacin D treatment of cervical cancer cells arrested the cell cycle in G1/S phase, inhibited constitutive expression of E6, Cyclin D1, CDK4, pRb, and Rb and induced the protein levels of p21 and p27. Cucurbitacin D also inhibited phosphorylation of STAT3 at Ser727 and Tyr705 residues as well as its downstream target genes c-Myc, and MMP9. Cucurbitacin D enhanced the expression of tumor suppressor microRNAs (miR-145, miRNA-143, and miRNA34a) in cervical cancer cells. Cucurbitacin D treatment (1 mg/kg body weight) effectively inhibited growth of cervical cancer cells derived orthotopic xenograft tumors in athymic nude mice. These results demonstrate the potential therapeutic efficacy of Cucurbitacin D against cervical cancer.

Development of polyvinylpyrrolidone/paclitaxel self-assemblies for breast cancer

The goal of this investigation was to develop and demonstrate a polymer/paclitaxel self-assembly (PTX-SA) formulation. Polymer/PTX-SAs were screened based on smaller size of formulation using dynamic light scattering analysis. Additionally, fluorescence microscopy and flow cytometry studies exhibited that polyvinylpyrrolidone (PVP)-based PTX-SAs (PVP/PTX-SAs) had superior cellular internalization capability in MCF7 and MDA-MB-231 breast cancer cells. The optimized PVP/PTX-SAs exhibited less toxicity to human red blood cells indicating a suitable formulation for reducing systemic toxicity. The formation of PVP and PTX self-assemblies was confirmed using fluorescence quenching and transmission electron microscopy which indicated that the PVP/PTX-SAs were spherical in shape with an average size range of 53.81 nm as detected by transmission electron microscopy (TEM). FTIR spectral analysis demonstrates incorporation of polymer and paclitaxel functional groups in PVP/PTX-SAs. Both proliferation (MTS) and clonogenic (colony formation) assays were used to validate superior anticancer activity of PVP/PTX-SAs in breast cancer cells over paclitaxel. Such superior anticancer activity was also demonstrated by downregulation of the expression of pro-survival protein (Bcl-xL), upregulation of apoptosis-associated proteins (Bid, Bax, cleaved caspase 7, and cleaved PARP) and β-tubulin stabilization. These results support the hypothesis that PVP/PTX-SAs improved paclitaxel delivery to cancer cells.

Ormeloxifene suppresses prostate tumor growth and metastatic phenotypes via inhibition of oncogenic β-catenin signaling and EMT progression

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Abstract 1312: PSMA antibody functionalized docetaxel-loaded magnetic nanoparticles for prostate cancer therapy

Objectives: Prostate cancer (PrCa) is the second most leading cause of cancer-related death in men in the United States. Chemotherapy (Docetaxel, Dox) is currently the most common first-line therapeutic option. However, adverse side effects and chemo-resistance of docetaxel limit its clinical use. Improving docetaxel targeted delivery and its activity at the tumor site using a targeted nanoparticle system could be an attractive strategy for PrCa therapy. Prostate Specific Membrane Antigen (PSMA) is highly overexpressed in PrCa cells, thus is a highly attractive molecular target for PrCa therapy. In this study, we developed and determined anti-cancer efficacy of a novel docetaxel loaded, PSMA targeted magnetic nanoparticle (PSMA-MNP-Dox) formulation for PrCa therapy. Methods: Docetaxel loaded magnetic nanoparticle (MNP-Dox) formulation is composed of an iron oxide core coated with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization). Therapeutic efficacy of this unique nanoparticle formulation was evaluated using clinically relevant cell line models (C4-2, PC-3, and DU-145) through cell proliferation and colony formation assays. Molecular effects of this formulation on apoptosis, anti-apotosis, and drug resistance associated proteins were evaluated using immunoblotting assays. Contrast imaging property of MNP-Dox formulation was examined using Phantom Gel MR imaging model. For active targeting, PSMA antibody conjugation to this formulation was achieved through N-hydroxysuccinimide group containing PEG polymer. Active targeting potential of this formulation was evaluated in PSMA+ (C4-2) and PSMA- (PC-3) cell lines, C4-2 generated tumor xenografts. Results: MNP-Dox formulation showed optimal particle size and zeta potential which can efficiently internalized in PrCa cells. Our formulation showed anti-cancer efficacy in prostate cancer cell lines. Additionally, it induces the expression of apoptosis associated proteins, Bax and Bad, cleaved PARP, and caspase 3, and downregulated the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Moreover, it also inhibited the expression of chemoresistance associated proteins (PSMA and MDR1). Our PSMA antibody targeted MNPs-Dox formulation exhibited a profound uptake pattern in PSMA+ cells (C4-2) compared to PSMA null (PC-3)- cells, suggesting its targeting potential. A similar targeting potential was also observed in ex-vivo studies while using C4-2 tumor xenografts, however, no intense targeting was observed in normal tissues due to lack of PSMA expression. Conclusion: PSMA antibody functionalized MNP-Dox formulation can efficiently target PSMA + PrCa cells and deliver docetaxel into prostate tumors. This targeted drug delivery system could reduce the dose of docetaxel required to kill cancer cells, thus minimizing long-term docetaxel associated systemic toxicity and drug-resistance. Citation Format: Prashanth Kumar Bhusetty Nagesh, Nia Johnson, Vijaya K.N. Boya, Pallabita Chowdhury, Aditya Ganju, Bilal Hafeez, Sheema Khan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. PSMA antibody functionalized docetaxel-loaded magnetic nanoparticles for prostate cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1312.

Tannic acid induces endoplasmic reticulum stress-mediated apoptosis in prostate cancer

Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G1 population, due to increase in p18INK4C and p21WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

Background:Cancer progression and metastasis is profoundly influenced by protein kinase D1 (PKD1) and metastasis-associated protein 1 (MTA1) in addition to other pathways. However, the nature of regulatory relationship between the PKD1 and MTA1, and its resulting impact on cancer metastasis remains unknown. Here we present evidence to establish that PKD1 is an upstream regulatory kinase of MTA1.Methods:Protein and mRNA expression of MTA1 in PKD1-overexpressing cells were determined using western blotting and reverse-transcription quantitative real-time PCR. Immunoprecipitation and proximity ligation assay (PLA) were used to determine the interaction between PKD1 and MTA1. PKD1-mediated nucleo-cytoplasmic export and polyubiquitin-dependent proteosomal degradation was determined using immunostaining. The correlation between PKD1 and MTA1 was determined using intra-tibial, subcutaneous xenograft, PTEN-knockout (PTEN-KO) and transgenic adenocarcinoma of mouse prostate (TRAMP) mouse models, as well as human cancer tissues.Results:We found that MTA1 is a PKD1-interacting substrate, and that PKD1 phosphorylates MTA1, supports its nucleus-to-cytoplasmic redistribution and utilises its N-terminal and kinase domains to effectively inhibit the levels of MTA1 via polyubiquitin-dependent proteosomal degradation. PKD1-mediated downregulation of MTA1 was accompanied by a significant suppression of prostate cancer progression and metastasis in physiologically relevant spontaneous tumour models. Accordingly, progression of human prostate tumours to increased invasiveness was also accompanied by decreased and increased levels of PKD1 and MTA1, respectively.Conclusions:Overall, this study, for the first time, establishes that PKD1 is an upstream regulatory kinase of MTA1 status and its associated metastatic activity, and that the PKD1-MTA1 axis could be targeted for anti-cancer strategies.

miRNA-205 Nanoformulation Sensitizes Prostate Cancer Cells to Chemotherapy

The therapeutic application of microRNA(s) in the field of cancer has generated significant attention in research. Previous studies have shown that miR-205 negatively regulates prostate cancer cell proliferation, metastasis, and drug resistance. However, the delivery of miR-205 is an unmet clinical need. Thus, the development of a viable nanoparticle platform to deliver miR-205 is highly sought. A novel magnetic nanoparticle (MNP)-based nanoplatform composed of an iron oxide core with poly(ethyleneimine)-poly(ethylene glycol) layer(s) was developed. An optimized nanoplatform composition was confirmed by examining the binding profiles of MNPs with miR-205 using agarose gel and fluorescence methods. The novel formulation was applied to prostate cancer cells for evaluating cellular uptake, miR-205 delivery, and anticancer, antimetastasis, and chemosensitization potentials against docetaxel treatment. The improved uptake and efficacy of formulations were studied with confocal imaging, flow cytometry, proliferation, clonogenicity, Western blot, q-RT-PCR, and chemosensitization assays. Our findings demonstrated that the miR-205 nanoplatform induces significant apoptosis and enhancing chemotherapeutic effects in prostate cancer cells. Overall, these study results provide a strong proof-of-concept for a novel nonviral-based nanoparticle protocol for effective microRNA delivery to prostate cancer cells.