Fibroblast growth factor receptor 1 inhibition suppresses pancreatic cancer chemoresistance and chemotherapy-driven aggressiveness.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: FGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse.

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Expression of fibroblast growth factor receptor 1 correlates inversely with the efficacy of single-agent fibroblast growth factor receptor-specific inhibitors in pancreatic cancer.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Highly Reproducible Quantitative Proteomics Analysis of Pancreatic Cancer Cells Reveals Proteome-Level Effects of a Novel Combination Drug Therapy That Induces Cancer Cell Death via Metabolic Remodeling and Activation of the Extrinsic Apoptosis Pathway.

Pancreatic cancer patients have poor survival rates and are frequently treated using gemcitabine (Gem). However, initial tumor sensitivity often gives way to rapid development of resistance. Gem-based drug combinations are employed to increase efficacy and mitigate resistance, but our understanding of molecular-level drug interactions, which could assist in the development of more effective therapeutic regimens, is limited. Global quantitative proteomic analysis could provide novel mechanistic insights into drug combination interactions, but it is challenging to achieve high-quality quantitative proteomics analysis of the large sample sets that are typically required for drug combination studies. Here, we investigated molecular-level temporal interactions of Gem with BGJ398 (infigratinib), a recently approved pan-FGFR inhibitor, in multiple treatment groups (N = 42 samples) using IonStar, a robust large-scale proteomics method that employs well-controlled, ultrahigh-resolution MS1 quantification. A total of 5514 proteins in the sample set were quantified without missing data, requiring >2 unique peptides/protein, <1% protein false discovery rate (FDR), <0.1% peptide FDR, and CV < 10%. Functional analysis of the differentially altered proteins revealed drug-dysregulated processes such as metabolism, apoptosis, and antigen presentation pathways. These changes were validated experimentally using Seahorse metabolic assays and immunoassays. Overall, in-depth analysis of large-scale proteomics data provided novel insights into possible mechanisms by which FGFR inhibitors complement and enhance Gem activity in pancreatic cancers.

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.

Synergistic Pharmacodynamic Effects of Gemcitabine and Fibroblast Growth Factor Receptor Inhibitors on Pancreatic Cancer Cell Cycle Kinetics and Proliferation.

Median survival of pancreatic ductal adenocarcinoma cancer (PDAC) is 6 months, with 9% 5-year survival. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and combination therapies integrating novel targeted agents could improve outcomes. Fibroblast growth factor (FGF) receptors (FGFRs) play important roles in PDAC growth and invasion. Therefore, FGFR inhibitors (FGFRi) merit further investigation. Efficacy of Gem combined with NVP-BGJ398, a pan-FGFRi, was investigated in multiple PDAC cell lines exposed to the drugs alone and combined. Cell cycle distribution and cell numbers were quantified over time. Two pharmacodynamic models were developed to investigate Gem/BGJ398 interactions quantitatively: a drug-mediated cell proliferation/death model, and a drug-perturbed cell cycle progression model. The models captured temporal changes in cell numbers, cell cycle progression, and cell death during drug exposure. Simultaneous fitting of all data provided reasonable parameter estimates. Therapeutic efficacy was then evaluated in a PDAC mouse model. Compared with Gem alone, combined Gem + FGFRi significantly downregulated ribonucleotide-diphosphate reductase large subunit 1 (RRM1), a gemcitabine resistance (GemR) biomarker, suggesting the FGFRi inhibited GemR emergence. The cell proliferation/death pharmacodynamic model estimated the drug interaction coefficient ψ death = 0.798, suggesting synergistic effects. The mechanism-based cell cycle progression model estimated drug interaction coefficient ψ cycle = 0.647, also suggesting synergy. Thus, FGFR inhibition appears to synergize with Gem in PDAC cells and tumors by sensitizing cells to Gem-mediated inhibition of proliferation and cell cycle progression. SIGNIFICANCE STATEMENT: An integrated approach of quantitative modeling and experimentation was employed to investigate the nature of fibroblast growth factor receptor inhibitor (FGFRi)/gemcitabine (Gem) interaction, and to identify mechanisms by which FGFRi exposure reverses Gem resistance in pancreatic cancer cells. The results show that FGFRi interacts synergistically with Gem to sensitize pancreatic cancer cells and tumors to Gem-mediated inhibition of proliferation and cell cycle progression. Thus, addition of FGFRi to standard-of-care Gem treatment could be a clinically deployable approach to enhance therapeutic benefit to pancreatic cancer patients.

Targeting Pyruvate Carboxylase by a Small Molecule Suppresses Breast Cancer Progression.

Rapid metabolism differentiates cancer cells from normal cells and relies on anaplerotic pathways. However, the mechanisms of anaplerosis-associated enzymes are rarely understood. The lack of potent and selective antimetabolism drugs restrains further clinical investigations. A small molecule ZY-444 ((N 4-((5-(4-(benzyloxy)phenyl)-2-thiophenyl)methyl)-N 2-isobutyl-2,4-pyrimidinediamine) is discovered to inhibit cancer cell proliferation specifically, having potent efficacies against tumor growth, metastasis, and recurrence. ZY-444 binds to cellular pyruvate carboxylase (PC), a key anaplerotic enzyme of the tricarboxylic acid cycle, and inactivates its catalytic activity. PC inhibition suppresses breast cancer growth and metastasis through inhibiting the Wnt/ß-catenin/Snail signaling pathway. Lower PC expression in patient tumors is correlated with significant survival benefits. Comparative profiles of PC expression in cancer versus normal tissues implicate the tumor selectivity of ZY-444. Overall, ZY-444 holds promise therapeutically as an anti-cancer metabolism agent.

Design and optimization of hybrid of 2,4-diaminopyrimidine and arylthiazole scaffold as anticancer cell proliferation and migration agents.

Therapeutics of metastatic or triple-negative breast cancer are still challenging in clinical. Herein we demonstrated the design and optimization of a series of hybrid of 2,4-diaminopyrimidine and arylthiazole derivatives for their anti-proliferative properties against two breast cancer cell lines (MCF-7 as human breast cancer and MDA-MB-231 as triple-negative breast cancer). More importantly, some of those compounds with potent antiproliferative activities also indicated excellent inhibitory activities against MDA-MB-231 cell migration. These results suggested that the new series of hybridation of aryl-thiazoles and aminopyrimidines could be identified and developed as novel highly potential anticancer agents against the triple-negative breast cancer as well as metastatic one in the future.

PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis.

The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin- and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ(-/-) mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ(-/-) mice induced fewer capillaries than in REGγ(+/+) littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ-PKA-FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases.

Synthesis and biological evaluation of novel tetrahydro-ß-carboline derivatives as antitumor growth and metastasis agents through inhibiting the transforming growth factor-ß signaling pathway.

The transforming growth factor beta (TGFß) signaling cascade is considered as one of the pivotal oncogenic pathways in most advanced cancers. Inhibition of the TGFß signaling pathway by specific antagonists, neutralizing antibodies, or small molecules is considered as an effective strategy for the treatment of tumor growth and metastasis. Here we demonstrated the identification of a series of tetrahydro-ß-carboline derivatives from virtual screening which potentially inhibit the TGFß signaling pathway. Optimization of the initial hit compound 2-benzoyl-1,3,4,9-tetrahydro-ß-carboline (8a) through substitution at different positions to define the structure-activity relationship resulted in the discovery of potent inhibitors of the TGFß signaling pathway. Among them, compound 8d, one of the tested compounds, not only showed potent inhibition of lung cancer cell proliferation and migration in vitro but also strongly suppressed growth of lung cancer and breast cancer in vivo.

Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2.

BACKGROUND AND PURPOSE: Angiogenesis-based therapy is an effective anti-tumour strategy and previous reports have shown some beneficial effects of a naturally occurring bioactive compound plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone). Here, we sought to determine the biological effects of plumbagin on signalling mechanisms during tumour angiogenesis. EXPERIMENTAL APPROACH: The effects of plumbagin were evaluated in various in vitro assays which utilised human umbilical vein endothelial cells (HUVEC) proliferation, migration and tube formation. Plumbagin was also evaluated in vivo using chicken embryo chorioallantoic membrane (CAM) and mouse corneal micropocket models., Human colon carcinoma and prostate cancer xenograft mouse models were used to evaluate the effects of plumbagin on angiogenesis. Immunofluorescence, GST pull-down and Western blotting were employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated Ras signalling pathways. KEY RESULTS: Plumbagin not only inhibited endothelial cell proliferation, migration and tube formation but also suppressed chicken chorioallantoic membrane neovascularzation and VEGF-induced mouse corneal angiogenesis. Moreover, plumbagin suppressed tumour angiogenesis and tumour growth in human colon carcinoma and prostate cancer xenograft mouse models. At a molecular level, plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signalling pathways mediated by VEGFR2 in HUVECs. CONCLUSIONS AND IMPLICATIONS: Plumbagin inhibited tumour angiogenesis and tumour growth by interference with the VEGFR2-mediated Ras signalling pathway in endothelial cells. Our findings demonstrate a molecular basis for the effects of plumbagin and suggest that this compound might have therapeutic ant-tumour effects.