👤 Tista Roy Chaudhuri

Pancreatic ductal adenocarcinoma (PDAC) is often intrinsically-resistant to standard-of-care chemotherapies such as gemcitabine. Acquired gemcitabine resistance (GemR) can arise from treatment of initially-sensitive tumors, and chemotherapy can increase tumor aggressiveness. We investigated the molecular mechanisms of chemoresistance and chemotherapy-driven tumor aggressiveness, which are understood incompletely. Differential proteomic analysis was employed to investigate chemotherapy-driven chemoresistance drivers and responses of PDAC cells and patient-derived tumor xenografts (PDX) having different chemosensitivities. We also investigated the prognostic value of FGFR1 expression in the efficacy of selective pan-FGFR inhibitor (FGFRi)-gemcitabine combinations. Quantitative proteomic analysis of a highly-GemR cell line revealed fibroblast growth factor receptor 1 (FGFR1) as the highest-expressed receptor tyrosine kinase. FGFR1 knockdown or FGFRi co-treatment enhanced gemcitabine efficacy and decreased GemR marker expression, implicating FGFR1 in augmentation of GemR. FGFRi treatment reduced PDX tumor progression and prolonged survival significantly, even in highly-resistant tumors in which neither single-agent showed efficacy. Gemcitabine exacerbated aggressiveness of highly-GemR tumors, based upon proliferation and metastatic markers. Combining FGFRi with gemcitabine or gemcitabine+nab-paclitaxel reversed tumor aggressiveness and progression, and prolonged survival significantly. In multiple PDAC PDXs, FGFR1 expression correlated with intrinsic tumor gemcitabine sensitivity. FGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse. Show less

Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them and devise approaches to stratify SHHi-responsive tumors noninvasively based on clinically-quantifiable parameters. Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intratumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key "nodes" responsible for EMT induction. Multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Nonresponding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification. Show less

Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. The mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. The pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. Single-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1. Show less

Aggregates of β-amyloid peptide are found to occur in brains of AD patients and are formed upon sequential cleavage of the amyloid precursor protein by BACE1 and γ-secretase. Strategies inhibiting either peptide aggregation or the rate limiting enzyme BACE1 have been in demand for its implication in AD therapeutics. The present study is undertaken to mine compounds with dual ability. In this context, some natural compounds that were already predicted as BACE1 inhibitors by our group, were further tested for their activity as aggregation inhibitors. A pharmacophore model built with known antiamyloidogenic compounds was then applied for screening the natural compounds previously predicted as BACE1 inhibitors. Subsequently experimental validation by Thioflavin-T and Aβ-GFP assay filtered four compounds genistein, syringetin, tamarixetin and ZINC53276039. Out of them, ZINC53276039 showed promising antiamyloidogenic activity to act as a potent inhibitor of aggregation. Interestingly, our previous study revealed syringetin and ZINC53276039 to be good BACE1 inhibitors while tamarixetin to be a moderate BACE1 inhibitor. These good to moderate BACE1 inhibitors with moderate to reasonable antiamyloidogenic activity might show potency in reducing the amyloid load of AD brains. Show less

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors. Show less

We have previously shown that the zinc finger transcription repressor SNAI2 (SLUG) represses tumor suppressor BRCA2-expression in non-dividing cells by binding to the E2-box upstream of the transcription start site. However, it is unclear how proliferating breast cancer (BC) cells that has higher oxidation state, overcome this repression. In this study, we provide insight into the mechanism of de-silencing of BRCA2 gene expression by PRDX5A, which is the longest member of the peroxiredoxin5 family, in proliferating breast cancer cells. We used cell synchronization and DNA affinity pulldown to analyze PRDX5A binding to the BRCA2 silencer. We used oxidative stress and microRNA (miRNA) treatments to study nuclear localization of PRDX5A and its impact on BRCA2-expression. We validated our findings using mutational, reporter assay, and immunofluorescence analyses. Under oxidative stress, proliferating BC cells express PRDX5 isoform A (PRDX5A). In the nucleus, PRDX5A binds to the BRCA2 silencer near the E2-box, displacing SLUG and enhancing BRCA2-expression. Nuclear PRDX5A is translated from the second AUG codon in frame to the first AUG codon in the PRDX5A transcript that retains all exons. Mutation of the first AUG increases nuclear localization of PRDX5A in MDA-MB-231 cells, but mutation of the second AUG decreases it. Increased mitronic hsa-miRNA-6855-3p levels under oxidative stress renders translation from the second AUG preferable. Mutational analysis using reporter assay uncovered a miR-6855-3p binding site between the first and second AUG codon in the PRDX5A transcript. miR-6855-3p mimic increases accumulation of nuclear PRDX5A and inhibits reporter gene translation. Oxidative stress increases miR-6855-3p expression and binding to the inter-AUG sequence of the PRDX5A transcript, promoting translation of nuclear PRDX5A. Nuclear PRDX5A relieves SLUG-mediated BRCA2 silencing, resulting in increased BRCA2-expression. Show less

Breast cancer, a heterogeneous disease has been broadly classified into oestrogen receptor positive (ER+) or oestrogen receptor negative (ER-) tumour types. Each of these tumours is dependent on specific signalling pathways for their progression. While high levels of survivin, an anti-apoptotic protein, increases aggressive behaviour in ER- breast tumours, oxidative stress (OS) promotes the progression of ER+ breast tumours. Mechanisms and molecular targets by which OS promotes tumourigenesis remain poorly understood. DETA-NONOate, a nitric oxide (NO)-donor induces OS in breast cancer cell lines by early re-localisation and downregulation of cellular survivin. Using in vivo models of HMLE(HRAS) xenografts and E2-induced breast tumours in ACI rats, we demonstrate that high OS downregulates survivin during initiation of tumourigenesis. Overexpression of survivin in HMLE(HRAS) cells led to a significant delay in tumour initiation and tumour volume in nude mice. This inverse relationship between survivin and OS was also observed in ER+ human breast tumours. We also demonstrate an upregulation of NADPH oxidase-1 (NOX1) and its activating protein p67, which are novel markers of OS in E2-induced tumours in ACI rats and as well as in ER+ human breast tumours. Our data, therefore, suggest that downregulation of survivin could be an important early event by which OS initiates breast tumour formation. Show less

We report evidence that cyclic-N(3) is exclusively produced in the 157-nm photolysis of ClN(3). Photoproduct translational energy measurements reveal a single-peaked distribution for an N(3)-formation channel with maximum and minimum translational energies matching the theoretically predicted minimum and maximum binding energies of cyclic-N(3), respectively. The absence of linear-N(3) greatly simplifies the data analysis. The zero-Kelvin heat of formation of cyclic-N(3) is derived experimentally (142+/-3.5 kcal/mol) and is in excellent agreement with the best existing determinations from other studies. Show less

We present results of near-threshold photoionization of N3 photofragments produced by laser photodissociation of ClN3 at 248 nm. The time of flight of recoiling N3 is used to resolve two photochemical channels producing N3, which exhibit different translational energy release. The two forms of N3 resolved in this way exhibit different photoionization thresholds, consistent with their assignment to linear (X 2pi(g)) and cyclic N3. This result agrees with the existing theoretical calculations of excited and ionic states of N3 and strengthens previous experimental results which suggested that the ClN3 photolysis produces a cyclic form of N3. Show less

We performed microarray gene expression analyses on the visual cortex of Old-World monkeys (Cercopithicus aethiops) in an effort to identify transcripts associated with developmental maturation and activity-driven changes during the visual critical period. Samples derived from normal animals and those subjected to monocular enucleation (ME) were hybridized to human Affymetrix HG-U95Av2 oligonucleotide microarrays (N = 12) and the results were independently validated by real-time quantitative RT-PCR. To identify genes exhibiting significant expression differences among our samples, the microarray hybridization data were processed with two software packages that use different analytical models (Affymetrix MicroArray Suite 5.0, dChip 1.2). We identified 108 transcripts within diverse functional categories that differed in their visual cortical expression at the height of the critical period when compared to adults. The expression levels of four transcripts were also globally modulated following ME during the critical period. These transcripts are particularly sensitive to ME during the critical period but are not significantly modulated in ME adults. Three of the ME-driven genes (NGFI-B, egr3, NARP) are known immediate-early genes (IEG) while the other (DUSP6) is a phosphatase that can regulate IEG expression. The putative biological significance of the ME-driven and developmentally regulated genes is discussed with respect to the critical period for activity-dependent visual cortical neuroplasticity. Show less