Novel Detection and Treatment Strategies for Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with an estimated 5-year survival rate of less than 9%. The high lethality of PDAC is due to two primary reasons: the discovery of PDAC at later stages, with locally invasive or metastatic disease present at the time of initial diagnosis as well as the lack of efficacious therapeutic interventions that significantly impact survival. In this dissertation, we sought to discover and test novel detection and treatment strategies for PDAC. Firstly, serum EVs were investigated as potential non-invasive liquid biopsy biomarkers, to serve as a means of early cancer detection. Secondly, a recently discovered form of cell death, ferroptosis, was investigated as a means of potentiating radiation therapy. The investigation into the potential of extracellular vesicles (EVs) as circulating biomarkers began with a label-free analysis of EVs via surface-enhanced Raman Spectroscopy (SERS) and principal component discriminant function analysis (PC-DFA), to identify tumor-specific spectral signatures. This method differentiated EVs originating from PDAC or normal pancreatic epithelial cell lines with 90% overall accuracy. The proof-of-concept application of this method to EVs purified from patient serum exhibited up to 87% and 90% predictive accuracy for healthy control and early PDAC individual samples, respectively. The specific EV surface proteins that may contribute to the observed SERS differences were investigated via surface shaving LC-MS/MS. This analysis provided protein targets that were selected and validated with a combination of bioinformatics, western blot, and immunogold labeling techniques. The first target protein selected for assessment via ELISA, EPHA2, showed elevated expression in complete cancer patient serum as compared to benign controls. Further, EV specific EPHA2 expression was capable of predicting cancer status in 25% (5/20) of the patient samples with 100% specificity. These data suggest a potential role of EV surface profiling for the early detection of PDAC. However, further work is required to increase the overall accuracy. Additionally, we sought to investigate the involvement of ferroptosis, in radiation-induced cell death. Ferroptosis is a non-apoptotic form of cell death that requires labile ferrous iron (Fe2+) and is caused by the reactive oxygen species (ROS) mediated build-up of lipid hydroperoxides. Further, we tested if the pharmaceutical induction of ferroptosis via the small molecule Erastin can potentiate the lethal effects of radiation in vitro and in vivo. We observed that radiation produces an increase in ROS and free Fe2+ leading to lipid hydroperoxidation, which was enhanced with the addition of Erastin culminating in the likely induction of ferroptosis. The combination of radiation and Erastin synergistically increased cell death in monocultures and patient-derived organoids as well as significantly reduced tumor size in xenograft mouse models. These findings suggest the potential of ferroptosis induction to improve radiation therapy, though specific mechanistic components require further evaluation. Therefore, further studies must be conducted to elucidate the specific role of ferroptosis in radiation-induced cell death. The combination of early detection and novel therapeutic intervention strategies offers a means of improving the survival of those with this dreaded disease

Trefoil Factor(s) and CA19.9: A Promising Panel for Early Detection of Pancreatic Cancer

BACKGROUND: Trefoil factors (TFF1, TFF2, and TFF3) are small secretory molecules that recently have gained significant attention in multiple studies as an integral component of pancreatic cancer (PC) subtype-specific gene signature. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 in combination with CA19.9 for detection of PC. METHODS: Trefoil factors (TFFs) gene expression was analyzed in publicly available cancer genome datasets, followed by assessment of their expression in genetically engineered spontaneous mouse model (GEM) of PC (KrasG12D; Pdx1-Cre (KC)) and in human tissue microarray consisting of normal pancreas adjacent to tumor (NAT), precursor lesions (PanIN), and various pathological grades of PC by immunohistochemistry (IHC). Serum TFFs and CA19.9 levels were evaluated via ELISA in comprehensive sample set (n = 362) comprised of independent training and validation sets each containing benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Univariate and multivariate logistic regression and receiver operating characteristic curves (ROC) were used to examine their diagnostic potential both alone and in combination with CA19.9. FINDINGS: The publicly available datasets and expression analysis revealed significant increased expression of TFF1, TFF2, and TFF3 in human PanINs and PC tissues. Assessment of KC mouse model also suggested upregulated expression of TFFs in PanIN lesions and early stage of PC. In serum analyses studies, TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to benign and CP control group while significant elevation in TFF3 levels were observed in CP group with no further elevation in its level in early stage PC group. In receiver operating curve (ROC) analyses, combination of TFFs with CA19.9 emerged as promising panel for discriminating early stage of PC (EPC) from BC (AUC INTERPRETATION: In silico, tissue and serum analyses validated significantly increased level of all TFFs in precursor lesions and early stages of PC. The combination of TFFs enhanced sensitivity and specificity of CA19.9 to discriminate early stage of PC from benign control and chronic pancreatitis groups

Disruption of FDPS/Rac1 Axis Radiosensitizes Pancreatic Ductal Adenocarcinoma by Attenuating DNA Damage Response and Immunosuppressive Signalling

BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient\u27s clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P \u3c 0.01 and P \u3c 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P \u3c 0.05, P \u3c 0.01, and P \u3c 0.001) and in vivo (P \u3c 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01)

ST6GalNAc-I Promotes Lung Cancer Metastasis by Altering MUC5AC Sialylation

Lung cancer (LC) is the leading cause of cancer-related mortality. However, the molecular mechanisms associated with the development of metastasis is poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models- KrasG12D ;Trp53R172H/+ ;Ad-Cre (KPA) and KrasG12D ; Ad-Cre (KA). Survival analysis showed significantly (P=0.0049) shorter survival in KPA tumor-bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of St6galnac-I in KPA compared to KA tumors. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid (SA) to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species-specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc-I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53R175H cells exhibited higher affinity towards STn. In addition, ST6GalNAc-I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc-I KO cells injected mice developed less liver metastasis (P=0.01) compared to controls, while co-localization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53R175H mediates ST6GalNAc-I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis

Contribution of Lipid Oxidation and Ferroptosis to Radiotherapy Efficacy

Radiotherapy promotes tumor cell death and senescence through the induction of oxidative damage. Recent work has highlighted the importance of lipid peroxidation for radiotherapy efficacy. Excessive lipid peroxidation can promote ferroptosis, a regulated form of cell death. In this review, we address the evidence supporting a role of ferroptosis in response to radiotherapy and discuss the molecular regulators that underlie this interaction. Finally, we postulate on the clinical implications for the intersection of ferroptosis and radiotherapy

Trefoil factor(s) and CA19.9: A promising panel for early detection of pancreatic cancerResearch in context

Background: Trefoil factors (TFF1, TFF2, and TFF3) are small secretory molecules that recently have gained significant attention in multiple studies as an integral component of pancreatic cancer (PC) subtype-specific gene signature. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 in combination with CA19.9 for detection of PC. Methods: Trefoil factors (TFFs) gene expression was analyzed in publicly available cancer genome datasets, followed by assessment of their expression in genetically engineered spontaneous mouse model (GEM) of PC (KrasG12D; Pdx1-Cre (KC)) and in human tissue microarray consisting of normal pancreas adjacent to tumor (NAT), precursor lesions (PanIN), and various pathological grades of PC by immunohistochemistry (IHC). Serum TFFs and CA19.9 levels were evaluated via ELISA in comprehensive sample set (n = 362) comprised of independent training and validation sets each containing benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Univariate and multivariate logistic regression and receiver operating characteristic curves (ROC) were used to examine their diagnostic potential both alone and in combination with CA19.9. Findings: The publicly available datasets and expression analysis revealed significant increased expression of TFF1, TFF2, and TFF3 in human PanINs and PC tissues. Assessment of KC mouse model also suggested upregulated expression of TFFs in PanIN lesions and early stage of PC. In serum analyses studies, TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to benign and CP control group while significant elevation in TFF3 levels were observed in CP group with no further elevation in its level in early stage PC group. In receiver operating curve (ROC) analyses, combination of TFFs with CA19.9 emerged as promising panel for discriminating early stage of PC (EPC) from BC (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93) as well as CP (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93). Notably, at 90% specificity (desired for blood-based biomarker panel), TFFs combination improved CA19.9 sensitivity by 10% and 25% to differentiate EPC from BC and CP respectively. In an independent blinded validation set, the combination of TFFs and CA19.9 (AUCTFF1+TFF2+TFF3+CA19.9 = 0.82) also improved the overall efficacy of CA19.9 (AUCCA19.9 = 0.66) to differentiate EPC from CP proving unique biomarker capabilities of TFFs to distinguish early stage of this deadly lethal disease. Interpretation: In silico, tissue and serum analyses validated significantly increased level of all TFFs in precursor lesions and early stages of PC. The combination of TFFs enhanced sensitivity and specificity of CA19.9 to discriminate early stage of PC from benign control and chronic pancreatitis groups. Keywords: Biomarker, Trefoil factor, TFF1, TFF2, TFF3, CA19.9, Pancreatic cance

Connectivity mapping-based identification of pharmacological inhibitor targeting HDAC6 in aggressive pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials

Substituent Effects Impact Surface Charge and Aggregation of Thiophenol-Labeled Gold Nanoparticles for SERS Biosensors

SERS immunoassay biosensors hold immense potential for clinical diagnostics due to their high sensitivity and growing interest in multi-marker panels. However, their development has been hindered by difficulties in designing compatible extrinsic Raman labels. Prior studies have largely focused on spectroscopic characteristics in selecting Raman reporter molecules (RRMs) for multiplexing since the presence of well-differentiated spectra is essential for simultaneous detection. However, these candidates often induce aggregation of the gold nanoparticles used as SERS nanotags despite their similarity to other effective RRMs. Thus, an improved understanding of factors affecting the aggregation of RRM-coated gold nanoparticles is needed. Substituent electronic effects on particle stability were investigated using various para-substituted thiophenols. The inductive and resonant effects of functional group modifications were strongly correlated with nanoparticle surface charge and hence their stability. Treatment with thiophenols diminished the negative surface charge of citrate-stabilized gold nanoparticles, but electron-withdrawing substituents limited the magnitude of this diminishment. It is proposed that this phenomenon arises by affecting the interplay of competing sulfur binding modes. This has wide-reaching implications for the design of biosensors using thiol-modified gold surfaces. A proof-of-concept multiplexed SERS biosensor was designed according to these findings using the two thiophenol compounds with the most electron-withdrawing substitutions: NO2 and CN