ABSTRACT
BACKGROUND: The Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ ß-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC. METHODS: Here, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated. RESULTS: High ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort. CONCLUSION: ROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/mortality , Pancreatic Neoplasms/mortality , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Biomarkers, Tumor/analysis , Carcinoma, Pancreatic Ductal/diagnosis , Carcinoma, Pancreatic Ductal/pathology , Datasets as Topic , Female , Humans , Male , Neoplasm Staging , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , Prognosis , Receptor Tyrosine Kinase-like Orphan Receptors/analysis , Wnt Signaling PathwayABSTRACT
AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BER-resistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCT-distal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites.
Subject(s)
Cytidine Deaminase/metabolism , DNA Mismatch Repair , DNA Repair , Mutation , Uracil-DNA Glycosidase/metabolism , Adoptive Transfer , Animals , Base Pairing , Base Sequence , Male , Mice, Inbred C57BL , Uracil/metabolism , Uracil-DNA Glycosidase/geneticsABSTRACT
Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition-fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (TUBB3/ßIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced ßIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors.
Subject(s)
Drug Delivery Systems , Nanoparticles/administration & dosage , Pancreatic Neoplasms/drug therapy , Polymers/chemistry , RNA, Small Interfering/genetics , Tubulin/chemistry , Animals , Cell Survival/drug effects , Humans , Methacrylates/chemistry , Mice , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/chemistry , Nylons/chemistry , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA, Small Interfering/administration & dosage , Tubulin/genetics , Tubulin/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Incidence of endometrial cancer (EC) is rising in the developed world. The current standard of care, hysterectomy, is often infeasible for younger patients and those with high body mass index. There are limited non-surgical treatment options and a lack of biologically relevant research models to investigate novel alternatives to surgery for EC. The aim of the present study was to develop a long-term, patient-derived explant (PDE) model of early-stage EC and demonstrate its use for investigating predictive biomarkers for a current non-surgical treatment option, the levonorgestrel intra-uterine system (LNG-IUS). Fresh tumour specimens were obtained from patients with early-stage endometrioid EC. Tumours were cut into explants, cultured on media-soaked gelatin sponges for up to 21 days and treated with LNG. Formalin-fixed, paraffin embedded (FFPE) blocks were generated for each explant after 21 days in culture. Tumour architecture and integrity were assessed by haematoxylin and eosin (H&E) and immunohistochemistry (IHC). IHC was additionally performed for the expression of five candidate biomarkers of LNG resistance. The developed ex vivo PDE model is capable of culturing explants from early-stage EC tumours long-term (21 Days). This model can complement existing models and may serve as a tool to validate results obtained in higher-throughput in vitro studies. Our study provides the foundation to validate the extent to which EC PDEs reflect patient response in future research.
Subject(s)
Endometrial Neoplasms , Intrauterine Devices, Medicated , Female , Humans , Levonorgestrel/pharmacology , Endometrial Neoplasms/pathology , Hysterectomy , BiomarkersABSTRACT
Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.
Subject(s)
Polymers/chemistry , RNA, Small Interfering/administration & dosage , Animals , Blotting, Western , Cell Line, Tumor , Chromatography, Gel , Female , Humans , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Electron, Transmission , Nanoparticles/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.
Subject(s)
Pancreatic Diseases , Pancreatic Neoplasms , Humans , Gemcitabine , Protein-Lysine 6-Oxidase , Pancreatic Neoplasms/drug therapyABSTRACT
Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP(+) cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.
Subject(s)
Adenine/chemistry , Deoxyuracil Nucleotides/metabolism , Genes, Immunoglobulin , Mutation , Thymine/chemistry , Animals , Base Pairing , Gene Expression , Germinal Center/metabolism , Humans , Mice , Mice, Inbred C57BL , Pyrophosphatases/metabolism , Retroviridae/genetics , Retroviridae/metabolismABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and no significant improvement in patient survival has been seen in the past three decades. Treatment options are limited and selection of chemotherapy in the clinic is usually based on the performance status of a patient rather than the biology of their disease. In recent years, research has attempted to unlock a personalised treatment strategy by identifying actionable molecular targets in tumour cells or using preclinical models to predict the effectiveness of chemotherapy. However, these approaches rely on the biology of PDAC tumour cells only and ignore the importance of the microenvironment and fibrotic stroma. In this review, we highlight the importance of the microenvironment in driving the chemoresistant nature of PDAC and the need for preclinical models to mimic the complex multi-cellular microenvironment of PDAC in the precision medicine pipeline. We discuss the potential for ex vivo whole-tissue culture models to inform precision medicine and their role in developing novel therapeutic strategies that hit both tumour and stromal compartments in PDAC. Thus, we highlight the critical role of the tumour microenvironment that needs to be addressed before a precision medicine program for PDAC can be implemented.
ABSTRACT
It was recently shown that it is possible to exploit the nanoparticle shape to selectively target endocytosis pathways found in cancer and not healthy cells. It is important to understand and compare the endocytosis pathways of nanoparticles in both cancer and healthy cells to restrict the healthy cells from taking up anticancer drugs to help reduce the side effects for patients. Here, the clathrin-mediated endocytosis inhibitor, hydroxychloroquine, and the anticancer drug, doxorubicin, are loaded into the same mesoporous silica nanorods. The use of nanorods was found to restrict the uptake by healthy cells but allowed cancer cells to take up the nanorods via the macropinocytosis pathway. Furthermore, it is shown that the nanorods can selectively deliver doxorubicin to the nucleus of breast cancer cells and to the cytoplasm of pancreatic cancer cells. The dual-drug-loaded nanorods were able to selectively kill the breast cancer cells in the presence of healthy breast cells. This study opens exciting possibilities of targeting cancer cells based on the material shape rather than targeting antibodies.
ABSTRACT
Interfacial cues in the tumor microenvironment direct the activity and assembly of multiple cell types. Pancreatic cancer, along with breast and prostate cancers, is enriched with cancer-associated fibroblasts (CAFs) that activate to coordinate the deposition of the extracellular matrix, which can comprise over 90% of the tumor mass. While it is clear that matrix underlies the severity of the disease, the relationship between stromal-tumor cell assembly and cell-matrix dynamics remains elusive. Micropatterned hydrogels deconstruct the interplay between matrix stiffness and geometric confinement, guiding heterotypic cell populations and matrix assembly in pancreatic cancer. Interfacial cues at the perimeter of microislands guide CAF migration and direct cancer cell assembly. Computational modeling shows curvature-stress dependent cellular localization for cancer and CAFs in coculture. Regions of convex curvature enhance edge stress that activates a myofibroblast phenotype in the CAFs with migration and increased collagen I deposition, ultimately leading to a central "corralling" of cancer cells. Inhibiting mechanotransduction pathways decreases CAF activation and the associated corralling phenotype. Together, this work reveals how interfacial biophysical cues underpin aspects of stromal desmoplasia, a hallmark of disease severity and chemoresistance in the pancreatic, breast, and prostate cancers, thereby providing a tool to expand stroma-targeting therapeutic strategies.
Subject(s)
Cancer-Associated Fibroblasts , Pancreatic Neoplasms , Coculture Techniques , Humans , Male , Mechanotransduction, Cellular , Stromal Cells , Tumor MicroenvironmentABSTRACT
The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues of human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 1-2 mm explants and cultured on gelatin sponges for 12 days. Immunohistochemistry revealed that human PDAC explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC explants were treated with Abraxane and we observed different levels of response between patients. PDAC explants were also transfected with polymeric nanoparticles + Cy5-siRNA and we observed abundant cytoplasmic distribution of Cy5-siRNA throughout the PDAC explants. Overall, our novel model retains the 3D architecture of human PDAC and has advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis, and no tissue manipulation, digestion, or artificial propagation of organoids. This provides unprecedented opportunity to study PDAC biology including tumour-stromal interactions and rapidly assess therapeutic response to drive personalised treatment.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Pancreatic Ductal/genetics , Cell Culture Techniques , Organoids/pathology , Adenocarcinoma/pathology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Extracellular Matrix/pathology , Extracellular Matrix/ultrastructure , Humans , Organoids/ultrastructure , Pancreas/pathology , Pancreas/ultrastructure , Tumor Microenvironment/geneticsABSTRACT
Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.
Subject(s)
Amino Acid Transport System y+/antagonists & inhibitors , Antibodies, Monoclonal/pharmacology , Cancer-Associated Fibroblasts/drug effects , Carcinoma, Pancreatic Ductal/prevention & control , Gene Expression Regulation, Neoplastic/drug effects , Pancreatic Neoplasms/prevention & control , Tumor Microenvironment , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/immunology , Animals , Apoptosis , Cancer-Associated Fibroblasts/immunology , Cancer-Associated Fibroblasts/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Pancreatic NeoplasmsABSTRACT
Pancreatic cancer is predicted to be the second leading cause of cancer-related death by 2025. The best chemotherapy only extends survival by an average of 18 weeks. The extensive fibrotic stroma surrounding the tumor curbs therapeutic options as chemotherapy drugs cannot freely penetrate the tumor. RNA interference (RNAi) has emerged as a promising approach to revolutionize cancer treatment. Small interfering RNA (siRNA) can be designed to inhibit the expression of any gene which is important given the high degree of genetic heterogeneity present in pancreatic tumors. Despite the potential of siRNA therapies, there are hurdles limiting their clinical application such as poor transport across biological barriers, limited cellular uptake, degradation, and rapid clearance. Nanotechnology can address these challenges. In fact, the past few decades have seen the conceptualization, design, pre-clinical testing and recent clinical approval of a RNAi nanodrug to treat disease. In this review, we comment on the current state of play of clinical trials evaluating siRNA nanodrugs and review pre-clinical studies investigating the efficacy of siRNA therapeutics in pancreatic cancer. We assess the physiological barriers unique to pancreatic cancer that need to be considered when designing and testing new nanomedicines for this disease.
Subject(s)
Nanoparticles , Pancreatic Neoplasms , Pharmaceutical Preparations , Gene Silencing , Humans , Nanomedicine , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , RNA Interference , RNA, Small Interfering/geneticsABSTRACT
Medulloblastoma is a malignant brain tumor diagnosed in children. Chemotherapy has improved survival rates to approximately 70%; however, children are often left with long-term treatment side effects. New therapies that maintain a high cure rate while reducing off-target toxicity are required. We describe for the first time the use of a bacteriophage-peptide display library to identify heptapeptides that bind to medulloblastoma cells. Two heptapeptides that demonstrated high [E1-3 (1)] or low [E1-7 (2)] medulloblastoma cell binding affinity were synthesized. The potential of the peptides to deliver a therapeutic drug to medulloblastoma cells with specificity was investigated by conjugating E1-3 (1) or E1-7 (2) to doxorubicin (5). Both peptide-drug conjugates were cytotoxic to medulloblastoma cells. E1-3 doxorubicin (3) could permeabilize an in vitro blood-brain barrier and showed a marked reduction in cytotoxicity compared to free doxorubicin (5) in nontumor cells. This study provides proof-of-concept for developing peptide-drug conjugates to inhibit medulloblastoma cell growth while minimizing off-target toxicity.
Subject(s)
Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Doxorubicin/administration & dosage , Drug Carriers/metabolism , Medulloblastoma/drug therapy , Oligopeptides/metabolism , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain Neoplasms/metabolism , Cell Line , Cell Line, Tumor , Child , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Drug Carriers/chemistry , Drug Delivery Systems , Humans , Medulloblastoma/metabolism , Oligopeptides/chemistry , Peptide LibraryABSTRACT
Staphylococcus aureus transcription factor QacR regulates expression of the qacA multidrug efflux determinant. In response to binding cationic lipophilic compounds, including ethidium and rhodamine 6G, QacR dissociates from the qacA operator alleviating repression. Such ligand binding uniformly induces a coil-to-helix transition of residues Thr(89)-Tyr(93) revealing an asymmetric binding pocket in QacR containing two distinct subpockets. Here, the functional significance of hydrophobic, aromatic, and polar residues characteristic of the rhodamine 6G pocket and the proximal Tyr(92), proposed to facilitate the transcriptionally active conformation, was examined. Notably, the presence of Tyr(92) was not essential for QacR structural changes between DNA-bound and induced conformations. Furthermore, although mutation of the majority of residues contacting rhodamine 6G exerted moderate effects on QacR-rhodamine 6G binding, mutation of Leu(54) and Gln(96), and cumulative mutations involving these with Tyr(93) and Tyr(123), imparted a dramatic decrease in QacR-rhodamine 6G binding affinity. This equated with impaired dissociation of QacR from its operator DNA in the presence of this ligand in S. aureus, delineating the important role of these residues in the QacR-rhodamine 6G interaction. Additionally, despite maintaining a high affinity for ethidium, QacR mutants involving Leu(54), Tyr(93), Gln(96), and Tyr(123), which denote the interface between the rhodamine 6G and ethidium subpockets, were unable to be induced from operator DNA in the presence of ethidium in S. aureus. This highlights the significant contribution of these residues to QacR-mediated derepression of qacA transcription following ligand binding in the distal subpocket and may be important for the general mechanism irrespective of the ligand bound.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Membrane Transport Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Staphylococcus aureus/genetics , Amino Acid Substitution , Binding Sites , Drug Resistance, Microbial , Ethidium/metabolism , Homeostasis , Ligands , Models, Molecular , Mutation , Protein Conformation , Repressor Proteins/genetics , Rhodamines/metabolism , Transcription, GeneticABSTRACT
Pancreatic cancer is a lethal malignancy which is refractory to most chemotherapy drugs. Recent landmark studies have shed new light on the complex genetic heterogeneity of pancreatic cancer and provide an opportunity to utilize "precision-based medicines" to target genes based on the genetic profile of an individual's tumor to increase the efficiency of chemotherapy and decrease tumor growth and metastases. Gene-silencing drugs in the form of short-interfering RNA (siRNA) have the potential to play an important role in precision medicine for pancreatic cancer by silencing the expression of genes including those considered difficult to inhibit (undruggable) using chemical agents. However, before siRNA can reach its clinical potential a delivery vehicle is needed to carry siRNA across the cell membrane and into the cytoplasm of the cell. Herein, we detail the methods required to use star polymer nanoparticles to deliver siRNA to pancreatic tumors in an orthotopic pancreatic cancer mouse model to silence the expression of an "undruggable" gene (ßIII-tubulin) that regulates pancreatic cancer growth and chemosensitivity.
Subject(s)
Drug Delivery Systems/methods , Nanoparticles/chemistry , Pancreatic Neoplasms/therapy , RNA, Small Interfering/genetics , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Silencing/drug effects , Humans , Mice , Nanoparticles/therapeutic use , Pancreatic Neoplasms/genetics , Polymers/chemistry , Polymers/pharmacology , RNA Interference/drug effects , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology , Tubulin/genetics , Tubulin Modulators/chemistry , Tubulin Modulators/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Patients with pancreatic ductal adenocarcinoma (PC) have a poor prognosis due to metastases and chemoresistance. PC is characterized by extensive fibrosis, which creates a hypoxic microenvironment, and leads to increased chemoresistance and intracellular oxidative stress. Thus, proteins that protect against oxidative stress are potential therapeutic targets for PC. A key protein that maintains genomic integrity against oxidative damage is MutY-Homolog (MYH). No prior studies have investigated the function of MYH in PC cells. Using siRNA, we showed that knockdown of MYH in PC cells 1) reduced PC cell proliferation and increased apoptosis; 2) further decreased PC cell growth in the presence of oxidative stress and chemotherapy agents (gemcitabine, paclitaxel and vincristine); 3) reduced PC cell metastatic potential; and 4) decreased PC tumor growth in a subcutaneous mouse model in vivo. The results from this study suggest MYH may be a novel therapeutic target for PC that could potentially improve patient outcome by reducing PC cell survival, increasing the efficacy of existing drugs and reducing metastatic spread.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Cell Proliferation/drug effects , DNA Glycosylases/metabolism , Pancreatic Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle/drug effects , Cell Line, Tumor , DNA Glycosylases/genetics , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred BALB C , Mice, Nude , Oxidative Stress/drug effects , Paclitaxel/pharmacology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA Interference , Signal Transduction/drug effects , Time Factors , Transfection , Tumor Burden/drug effects , Tumor Hypoxia , Tumor Microenvironment , Vincristine/pharmacology , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is a highly chemoresistant and metastatic disease with a dismal 5-year survival rate of 6%. More effective therapeutic targets and approaches are urgently needed to tackle this devastating disease. The base excision repair (BER) pathway has been identified as a predictor of therapeutic response, prognostic factor, and therapeutic target in a variety of cancers. This review will discuss our current understanding of BER in PDA and its potential to improve PDA treatment.
ABSTRACT
Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, ß-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of ß-tubulins in pancreatic cancer are unknown. We measured the expression of different ß-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence ßIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of ßIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that ßIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing ßIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of ßIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that ßIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients.