Tumor Progression In Pancreatic Cancer Research Articles

PITX2 functions as a transcription factor for GPX4 and protects pancreatic cancer cells from ferroptosis

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.

99mTc]Tc-labeled cyc-DX600-HYNIC as a SPECT probe for ACE2-specific pancreatic cancer imaging.

As a regulator in renin-angiotensin-aldosterone system, angiotensin-converting enzyme 2 (ACE2) closely correlated with tumor progression of pancreatic cancer, meantime, was easily affected by a variety of factors. [99mTc]Tc-cyc-DX600 SPECT was established as an ACE2-specific imaging protocol to figure out the ACE2 status in pancreatic tumor. BALB/C-NU mice were used to prepare the subcutaneous cell derived xenograft (CDX) models with HEK-293T or HEK-293T/hACE2 cells to validate ACE2 specificity of [99mTc]Tc-cyc-DX600 SPECT and establish SPECT imaging protocol. On the basis of [99mTc]Tc-cyc-DX600 SPECT and [18F]F-FDG PET/CT, ACE2-dependence on tumor size and tumor metabolism were further verified on orthotopic pancreatic cancer model with KPC cells. Immunohistochemical analysis was used to demonstrate the findings on ACE2 SPECT. [99mTc]Tc-cyc-DX600 was of superior tumor uptake in HEK-293T/hACE2 CDX than wild type (6.74 ± 0.31 %ID/mL vs 1.83 ± 0.26 %ID/mL at 1.5 h post injection (p.i.); 3.14 ± 0.31 %ID/mL vs 1.16 ± 0.15 %ID/mL at 4.5 h p.i.). For the CDX models with PANC-1 cells, a significant negative correlation between the slope of tumor volume and tumor uptake was observed (r = -0.382 for the 1-4th day; r = -0.146 for the 1-5th day; r = -0.114 for the 1-6th day; r = -0.152 for the 1-7th day; but P > 0.05 for all). For orthotopic pancreatic cancer model, the linear correlation between FDG PET and ACE2 SPECT of the pancreatic lesions was negative (r = -0.878), the quantitative values of ACE2 SPCET was positively correlated with the volume of primary lesions (r = 0.752) and also positively correlated with the quantitative values of ACE2 immunohistochemical analysis (r = 0.991). Conclusively, [99mTc]Tc-cyc-DX600 SPECT is an ACE2-specific imaging protocol with clinical translational potential, adding multidimensional information on the disease progression of pancreatic cancer.

Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

ABSTRACT MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) orchestrates diverse environmental signals to facilitate cell growth and is frequently activated in cancer. Translocation of MTORC1 from the cytosol to the lysosomal surface by the RRAG GTPases is the key step in MTORC1 activation. Here, we demonstrated that transcription factors MEF2A and MEF2D synergistically regulated MTORC1 activation via modulating its cyto-lysosome shutting. Mechanically, MEF2A and MEF2D controlled the transcription of FNIP1 and FNIP2, the components of the FLCN-FNIP1 or FNIP2 complex that acts as a RRAGC-RRAGD GTPase-activating element to promote the recruitment of MTORC1 to lysosome and its activation. Furthermore, we determined that the pro-oncogenic protein kinase SRC/c-Src directly phosphorylated MEF2D at three conserved tyrosine residues. The tyrosine phosphorylation enhanced MEF2D transcriptional activity and was indispensable for MTORC1 activation. Finally, both the protein and tyrosine phosphorylation levels of MEF2D are elevated in human pancreatic cancers, positively correlating with MTORC1 activity. Depletion of both MEF2A and MEF2D or expressing the unphosphorylatable MEF2D mutant suppressed tumor cell growth. Thus, our study revealed a transcriptional regulatory mechanism of MTORC1 that promoted cell anabolism and proliferation and uncovered its critical role in pancreatic cancer progression. Abbreviation: ACTB: actin beta; ChIP: chromatin immunoprecipitation; EGF: epidermal growth factor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FLCN: folliculin; FNIP1: folliculin interacting protein 1; FNIP2: folliculin interacting protein 2; GAP: GTPase activator protein; GEF: guanine nucleotide exchange factors; GTPase: guanosine triphosphatase; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF2: myocyte enhancer factor 2; MEF2A: myocyte enhancer factor 2A; MEF2D: myocyte enhancer factor 2D; MEF2D-3YF: Y131F, Y333F, Y337F mutant; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NR4A1: nuclear receptor subfamily 4 group A member 1; RPTOR: regulatory associated protein of MTOR complex 1; RHEB: Ras homolog, mTORC1 binding; RPS6KB1: ribosomal protein S6 kinase B1; RRAG: Ras related GTP binding; RT-qPCR: real time-quantitative PCR; SRC: SRC proto-oncogene, non-receptor tyrosine kinase; TMEM192: transmembrane protein 192; WT: wild-type.

CCR4 Blockade Diminishes Intratumoral Macrophage Recruitment and Augments Survival of Syngeneic Pancreatic Cancer-Bearing Mice.

Pancreatic cancer is known for its tumor microenvironment (TME), which is rich in stromal and immune cells supporting cancer growth and therapy resistance. In particular, tumor-associated macrophages (TAMs) are known for their angiogenesis- and metastasis-promoting properties, which lead to the failure of conventional therapies for pancreatic cancer. Hence, treatment options targeting TAMs are needed. The C-C chemokine receptor type 4 (CCR4) is critical for immune cell recruitment into the TME, and in this paper we explore the effects of its genetic or immunotherapeutic blockade in pancreatic-cancer-bearing mice. Murine PDA6606 pancreatic cancer cells and murine peritoneal macrophages were used for in vitro migration assays. In vivo, a syngeneic, orthotropic pancreatic cancer model was established. Tumor growth and survival were monitored under prophylactic and therapeutic application of a CCR4 antagonist (AF-399/420/18025) in wildtype (CCR4wt) and CCR4-knockout (CCR4-/-) mice. Immune infiltration was monitored in tumor tissue sections and via flow cytometry of lysed tumors. PDA6606 cells induced less migration in CCR4-/- than in CCR4wt macrophages in vitro. Pancreatic TAM infiltration was higher, and survival was reduced in CCR4wt mice compared to CCR4-/- mice. Antagonizing CCR4 in wildtype mice revealed similar results as in CCR4-/- mice without antagonization. Prophylactic CCR4 antagonist application in wildtype mice was more efficient than therapeutic antagonization. CCR4 seems to be critically involved in TAM generation and tumor progression in pancreatic cancer. CCR4 blockade may help prolong the relapse-free period after curative surgery in pancreatic cancer and improve prognosis.

COL12A1 Acts as a Novel Prognosis Biomarker and Activates Cancer-Associated Fibroblasts in Pancreatic Cancer through Bioinformatics and Experimental Validation

Pancreatic cancer remains one of the most challenging malignancies to date and is associated with poor survival. Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Herein, we report our discoveries in this research area. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. Survival and COX regression analyses revealed the significant clinical prognostic value of COL12A1 expression in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. This was verified with our PCR analysis in cancer cells and CAFs. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers actin alpha 2 (ACTA2), fibroblast activation protein (FAP), and fibroblast-specific protein 1 (FSP1). Meanwhile, the interleukin 6 (IL6), CXC chemokine Ligand-5 (CXCL5), and CXC chemokine Ligand-10 (CXCL10) expressions were inhibited, and the cancer-promoting effect was reversed by COL12A1 knockdown. Therefore, we demonstrated the potential prognostic and target therapy value of COL12A1 expression in pancreatic cancer and elucidated the molecular mechanism underlying its role in CAFs. The findings of this study might provide new opportunities for TME-targeted therapies in pancreatic cancer.

MiR-4299 inhibits tumor progression in pancreatic cancer through targeting ADAM17.

Pancreatic cancer (PC) is one of the most aggressive malignant tumors in human beings. Tumor capacity of evading immune-mediated lysis is a critical step in PC malignant progression. We aimed to evaluate the underlying regulatory mechanism of miR-4299 in the proliferation, metastasis, apoptosis, and immune escape in PC. miR-4299 and ADAM17 expressions in PC tissues and cell lines were detected using qRT-PCR. MTT assay and flow cytometry were used to detect cell viability and apoptosis, respectively. A luciferase reporter gene assay was conducted to confirm the targeted relationship between miR-4299 and ADAM17. Xenograft tumors in nude mice were used to detect tumorigenesis in vivo. PC cells were co-cultured with NK cells for determining the immune escape ability. NKG2D-positive rate of NK cells was detected using flow cytometry; NK cell-killing ability was detected using MTT assay. miR-4299 was downregulated in PC tissues and cell lines. miR-4299 inhibited PC cell proliferation and invasion, promoted cell apoptosis, and reduced PC tumor growth in vivo. ADAM17 3'UTR directly bound to miR-4299. ADAM17 overexpression could reverse miR-4299 effects on PC cell viability, invasion, apoptosis, and immune escape. miR-4299 exerted suppressive effects on PC cell proliferation, invasion, and immune escape via targeting ADAM17 expression. This study revealed a novel miR-4299/ADAM17 axis-modulating PC progression and proposed to concern the immune regulatory mechanism of miRNAs in PC development.

Identification of a Novel Risk Model: A Five-Gene Prognostic Signature for Pancreatic Cancer.

Objective Biomarkers for pancreatic cancer (PCa) prognosis provide evidence for improving the survival outcome of this disease. This study aimed to identify a prognostic risk model based on gene expression profiling of microarray bioinformatics analysis. Methods Prognostic immune genes in the TCGA-PAAD cohort were identified using the univariate Cox regression and Kaplan–Meier survival analysis. Multivariate Cox regression (stepAIC) was used to identify prognostic genes from the top 20 hub genes in the protein-protein interaction (PPI) network. A prognostic risk model was established and its performance in predicting the overall survival in PCa was validated in GSE62452. Gene mutations and infiltration immune cells in PCa tumors were analyzed using online databases. Results Univariate Cox regression and Kaplan–Meier survival analyses identified 128 prognostic genes. Multivariate Cox regression (stepAIC) identified five prognostic genes (PLCG1, MET, TNFSF10, CXCL9, and TLR3) out of the 20 hub genes in the PPI network. A prognostic risk model was established using the signature of five genes. This model had moderate to high accuracies (AUC > 0.700) in predicting 3-year and 5-year overall survival in TCGA and GSE62452 cohorts. The Kaplan–Meier survival analysis showed that high-risk scores were correlated with poor survival outcomes in PCa (p < 0.05). Also, mutations in the five genes were related to poor survival. The five genes were related to multiple immune cells. Conclusions The prognostic risk model was significantly correlated with the survival in PCa patients. This model modulated PCa tumor progression and prognosis by regulating immune cell infiltration.

Combined Inhibition of FOSL-1 and YAP Using siRNA-Lipoplexes Reduces the Growth of Pancreatic Tumor

Simple SummaryIntercepting the molecular mechanisms implicated in pancreatic cancer progression can be an efficient therapeutic approach to treat this aggressive tumor. The Hippo pathway is a key mechanism driving tumor progression, even in the absence of KRAS activation. When this pathway is switched off, the transcriptional coactivator YAP is translocated into the nucleus and induces the activation of several genes implicated in tumor progression and apoptosis inhibition. FOSL-1 is a transcription factor that synergizes with YAP, forming a transcriptional complex. It has been shown to have a good therapeutic outcome when they are individually inhibited. In this work, we showed for the first time that the combined inhibition of YAP and FOSL-1 mRNA expression, using siRNA-lipoplexes, induces superior control over tumor growth in vitro and in vivo, compared to the individual treatments, and a reduction of the tumor stroma. The results offer a new therapeutic approach for pancreatic cancer treatment.Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer.

RhoGDI2 induced malignant phenotypes of pancreatic cancer cells via regulating Snail expression.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been shown to contribute to the aggressive phenotypes of human cancers, such as tumor metastasis and chemoresistance. This study aimed to assess the effects of RhoGDI2 on tumor progression and chemoresistance in pancreatic cancer cells. The expression of RhoGDI2 in pancreatic cancer cells was detected by Western blot analysis. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. The correlation between RhoGDI2 and Snail was also analyzed. Differential expression of RhoGDI2 protein in pancreatic cancer cell lines was identified. Gain-of-function and loss-of-function experiments showed that RhoGDI2 induced the malignant phenotypes of pancreatic cancer cells, including proliferation, migration, invasion, and gemcitabine (GEM) chemoresistance. The upregulation of RhoGDI2 stimulated the expression of Snail, resulting in the altered expression of epithelial marker E-cadherin and mesenchymal marker Vimentin, which were characteristics of the tumorigenic activity of epithelial-mesenchymal transition. The expression of RhoGDI2 and Snail was upregulated in clinical tumor samples, and higher expression of RhoGDI2 or Snail was significantly associated with poor patient survival in pancreatic ductal adenocarcinoma (PDAC). The findings indicated that RhoGDI2 promoted GEM resistance and tumor progression in pancreatic cancer and that RhoGDI2 might be a potential therapeutic target in patients with PDAC.

MiR-135b-5p is an oncogene in pancreatic cancer to regulate GPRC5A expression by targeting transcription factor KLF4

KLF4 is implicated in tumor progression of pancreatic cancer, but the molecular regulatory mechanism of KLF4 needs to be further specified. We aimed to probe molecular regulatory mechanism of KLF4 in malignant progression of pancreatic cancer. qRT-PCR or western blot was completed to test levels of predicted genes. Dual-luciferase and chromatin immunoprecipitation (ChIP) assays were designed to validate binding between genes. Cell viability and oncogenicity detection were used for in vitro and vivo functional assessment. KLF4 was a downstream target of miR-135b-5p. KLF4 could regulate GPRC5A level. MiR-135b-5p was notably increased in cancer cells, and overexpressing KLF4 functioned a tumor repressive role, which could be restored by miR-135b-5p. Besides, cell malignant phenotypes could be inhibited through reducing miR-135b-5p level, but they were restored by GPRC5A. Our results stressed that KLF4, as a vital target of miR-135b-5p, could influence promoter region of GPRC5A, thus affecting the malignant progression of pancreatic cancer.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

Simple SummaryA feature of pancreatic cancer (PC) is the frequent overexpression of tyrosine kinase membrane receptor HER2 along with its membrane partner the MUC4 oncomucin in the early stages of the pancreatic carcinogenesis. However, therapeutic approaches targeting HER2 in PC are not efficient. MUC4 could indeed represent an alternative therapeutic strategy to target HER2 signaling pathway, but this approach needs to characterize MUC4/HER2 interaction at the molecular level. In this study, we successfully showed the impact of the EGF domains of MUC4 on HER2 binding affinity and demonstrated their “growth factor-like” biological activities in PC cells. Moreover, homology models of the MUC4EGF/HER2 complexes allowed identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results allow a better understanding of the mechanisms involved in the MUC4/HER2 complex formation and may lead to the design of potential MUC4/HER2 inhibitors.The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their “growth factor-like” biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and β-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

Abstract PO-038: LAMC2: New player in stemness and tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating and essentially incurable disease, typically characterized by high chemoresistance and metastatic spread attributable to cancer stem cells (CSCs). This subpopulation is critical for tumor initiation and recurrence, but the mechanism through which they acquire metastatic traits is not well understood. Hence, targeting the CSC niche and their plasticity could be a complementary therapeutic strategy against cancer. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein, which controls cell motility and adhesion and is widely expressed in the majority of human tumors. However, its role in PDAC remains largely unknown. In several patient cohorts we observed that high levels of LAMC2 significantly correlated with shorter overall survival. In addition, the tissue microarray analysis on PDAC sections revealed prognostic significance of LAMC2 expression in tumor with high grade of aggressiveness (i.e., G2 and G3). To determine the role of LAMC2 in sustaining tumorigenicity, we knocked down it in patient-derived xenografts (PDX) cells using lentiviral shRNA constructs. The silencing of LAMC2 resulted in decreased self-renewal, invasiveness, tumorigenicity and gemcitabine resistance both in vitro and in vivo. To identify or track the LAMC2 tumor cell population in an intact environment we engineered primary PDAC cells that carry EGFP cassette knocked in the LAMC2 locus through the CRISPR-Cas9 technique. Analysis of LAMC2-EGFP+ cells isolated from tumor demonstrated that these cells express a gene program similar to that of highly metastatic stem cells and that they initiate and propagate both the primary tumor and the metastasis to recipient mice very efficiently compared to their counterpart. In conclusion, we identified a highly metastatic subpopulation of cancer stem cells, characterized by high levels of LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in combination with conventional therapy. Citation Format: Donatella Delle Cave, Tea Teresa Iavazzo, Maria Mangini, Gennaro Andolfi, Teresa Pirozzi, Annalisa Di Domenico, Annachiara De Luca, Enza Lonardo. LAMC2: New player in stemness and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-038.

Analysis of KRAS Mutation Subtype in Tissue DNA and Cell-Free DNA Using Droplet Digital PCR and the Function of Cell-Free DNA as a Recurrence Predictive Marker in Pancreatic Cancer.

KRAS mutation is a major regulator in the tumor progression of pancreatic cancer. Here, we compared the frequency and mutation burden of KRAS mutation subtypes with paired tumor tissue and blood in patients and examined their clinical significance. DNA from tumor tissues and cell-free DNA (cfDNA) from preoperative blood were obtained from 70 patients with pancreatic cancer. Subtypes and mutation burdens of KRAS G12D and G12V mutations were evaluated using droplet digital PCR. Comparing the presence of mutations in tissue, accumulative and simultaneous mutations of G12D or G12V were identified of 67 (95.7%), and 48 patients (68.6%). Conversely, in blood, they were only identified in 18 (25.7%) and four (5.7%) patients; respectively. Next, comparing the mutation burden in tissue, the mutation burden varied from less than 0.1 to more than five, whereas that of cfDNA in blood was mostly between one and five, as cases with a mutation burden lower than 0.1 and higher than five were rare. Finally, the presence of the G12V mutation alone in cfDNA and the combination of the G12V mutation with elevated CA 19-9 levels were associated with poor recurrence-free survival. These fundamental data on the KRAS mutation subtypes and their clinical significance could support their potential as predictive markers for postoperative recurrence.

Long noncoding RNA LINC00261 upregulates ITIH5 to impair tumorigenic ability of pancreatic cancer stem cells

Long noncoding RNAs (lncRNAs) are implicated tumor development in a range of different cancers, including pancreatic cancer (PC). Cancer stem cells (CSCs), a drug-resistant cancer cell subset, drive tumor progression in PC. In this work, we aimed to investigate the mechanism by which lncRNA LINC00261 affects the biological functions of CSCs during the progression of PC. Microarray analysis of differentially expressed genes and lncRNAs suggested that LINC00261 is downregulated in PC. Both LINC00261 and ITIH5 were confirmed to be downregulated in PC cells and PC stem cells. Gain-of-function and loss-of-function investigations were performed to analyze their effects on cell proliferation, drug resistance, cell cycle distribution, self-renewal, invasion, and ultimately overall tumorigenicity. These experiments revealed that the expression of stem cell markers was reduced, and cell proliferation, self-renewal ability, cell invasion, drug resistance, and tumorigenicity were all suppressed by upregulation of LINC00261 or ITIH5. The results of dual-luciferase reporter gene, ChIP, and RIP assays indicated that LINC00261 binds directly to GATA6, increasing its activity at the ITIH5 promoter. The presence of LINC00261 and GATA6 inhibited the self-renewal and tumorigenesis of PC stem cells, while silence of ITIH5 rescued those functions. Collectively, this study identifies the tumor suppressive activity of LINC00261 in PC, showing that this lncRNA limits the functions of PC stem through an ITIH5/GATA6 regulatory pathway.

Notch signaling inhibitor and anti-PD-L1 antibody combination therapies decelerate tumor progression in pancreatic cancer

Abstract Objective: Pancreatic cancer (PC) is a highly lethal malignancy with an immunosuppressive environment. Yet, current immune checkpoint inhibitor monotherapies have shown limited efficacy in PC, prompting the need for combination therapies. Herein, we hypothesized that combinations of Notch signaling inhibitor and anti-ligand programmed death-ligand 1 (PD-L1) antibody immunotherapy would show synergistic efficacy. Methods: The baseline expression of PD-L1 and HES1 was measured in PC cell lines, single-cell RNA-seq data of PC (GSA: CRA001160), and cBioPortal databases. In an in vitro study, MIA PaCa2 and SW1990 were used to explore the mechanism between Notch signaling and PD-L1. To study the effects in vivo, a subcutaneous tumor model was established using Pan02 cells treated with either anti-PD-L1 monoclonal antibody and/or Notch inhibitor DAPT. The study performed involving human samples was approved by the Ethics Committee of Peking Union Medical College Hospital (approval No. S-K460, approval date: April 23, 2018). Animal studies were approved by the Animal Research Ethics Committee of Peking Union Medical College Hospital (approval No. XHDW-2019-049, approval date: November 28, 2019). Results: The Notch signaling inhibitor upregulated PD-L1 expression in PC tumor cells both in vitro and in vivo. Notch effector HES1 knockdown produced PD-L1 upregulation in both MIA PaCa2 and SW1990 cells. Combined DAPT and anti-PD-L1 antibody treatment of Pan02 subcutaneous tumor model resulted in significantly reduced tumor weights compared to that with monotherapy, as well as significantly reduced Ki67 than that in the monotherapy group and control group. Flow cytometry analysis revealed significantly increased CD8+ T cell infiltration in tumors of the combination group compared with those of the monotherapy group. Conclusion: Notch signaling blockade might enhance the antitumor effect of anti-PD-L1 therapy in PC.

Safety of perioperative hyperthermic intraperitoneal chemotherapy with gemcitabine in patients with resected pancreatic adenocarcinoma: a pilot study of the clinical trial EudraCT 2016-004298-41.

Hyperthermic intraperitoneal chemotherapy (HIPEC) with gemcitabine, after cytoreductive surgery, may reduce the tumor progression of pancreatic cancer through the reduction of the neoplastic volume and the subpopulation of residual pancreatic cancer stem cells, improving the survival of patients with pancreatic cancer and decreasing the recurrence of the disease. A pilot study is performed with the first ten patients in the experimental group. A randomized study (phase II-III clinical trial) that requires a population of 42 patients, with 21 patients in each group. All patients have a diagnosis of ductal adenocarcinoma of the pancreas, which will be surgically resected with curative intention. (I) Group I: after an R0 resection, patients receive individualized adjuvant treatments. (II) Group II: after an R0 resection, HIPEC is performed with gemcitabine (120 mg/m2 for 30 min), and they also receive individualized adjuvant treatments. To analyze the safety of the procedure, the main variables measured were as follows: grades of complications by means of the Clavien-Dindo system: pancreas surgery complications (e.g., pancreatic fistula, perioperative hemorrhage, delayed gastric emptying, biliary fistula), operative mortality, and laboratory parameters to control system functions. Values were measured three times: preoperatively, twenty-four hours after surgery, and on the 7th postoperative day. From 2018 to 2019, 31 patients were recruited for our clinical trial. Fifteen patients were excluded because of intraoperative unresectability or a different intraoperative histologic diagnosis. Ten patients were included in the experimental group (resection plus HIPEC gemcitabine). The mean age was 65±7 years, and six patients were female (60%). We confirmed the histologic diagnosis of ductal pancreatic adenocarcinoma in all patients prior to HIPEC. Total pancreatectomy was performed in five patients. The surgical median time was 360 min, and the hospital stay was 11 days. Four patients showed complications classified as Clavien-Dindo type II and one showing type I. Six patients were classified as having stage III tumors. To date, no hospital mortality, locoregional recurrence, or differences between the two groups in terms of perioperative complications, biochemical and gasometric values, or Clavien-Dindo complication grades were observed. Our clinical pilot study demonstrated a similar perioperative outcome that allows the trial until main objectives are achieved.

Abstract PO-054: A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types, with more than 90% of patients succumbing to the disease within five years of diagnosis. The most notable hallmark of PDAC tumors is an inordinately dense stromal reaction comprised of extracellular matrix proteins, immune infiltrates, endothelial cells, and activated fibroblasts. The stroma experiences a dramatic expansion in parallel with the progression of the tumor, suggesting it is an active participant in PDAC development. However, key studies targeting stromal development through Hedgehog signaling revealed tumor-suppressive functions of the stroma, further complicating the strategy to target the stroma as a single component. Thus, our understanding of the complex relationship between the tumor and its microenvironment and how to effectively target the stroma itself remains incomplete. Previous work by our lab demonstrates that phosphatase and tensin homolog (PTEN) expression is lost in approximately 25% of patients in a PDAC cohort, and lower PTEN expression correlates with worsened patient outcome. Additionally, inhibition of Hedgehog signaling results in loss of PTEN expression and subsequent activation of Akt signaling pathways, and increased tumor growth. The purpose of the current study is to determine the effect of PTEN loss in PDAC cancer-associated fibroblasts (CAFs) on tumor progression and dissemination, and immune evasion. Using a combination of in vitro and in vivo experiments, we have found robust activation of Signal Transducer and Activator of Transcription 3 (STAT3) in PTEN-null pancreatic fibroblasts. We also see selective alterations in the secretome of pancreatic fibroblasts, including a marked increase in IL-6, CXCL1, and CXCL2. Our group has developed a genetically engineered mouse model of PDAC using a dual recombinase system in which the fibroblast-specific protein-1 (FspCre) transgene is used to conditionally delete genes of interest in the fibroblasts of Pdx1Flpki/+;FSF-KrasG12D/+;p53frt/+ (KPF) mice while driving tumorigenesis in the pancreatic epithelium. Conditional deletion of STAT3 in stromal fibroblasts of these mice results in increased overall survival, reduced tumor progression, and alterations within the immune landscape of the tumor microenvironment. Based on our current findings, we believe that a PTEN/STAT3/CXCL1/2 signaling axis within stromal CAFs produces an immunosuppressive tumor microenvironment that favors PDAC progression. Future experiments are needed to validate and confirm this conclusion, including defining the exact mechanisms by which PTEN loss leads to STAT3 activation and elevated secretion of CXCL1/2. This project further highlights the nuanced and complex relationship between PDAC tumor and stroma, and may serve to advance our understanding of how the stroma can be more precisely and effectively targeted to reduce tumor burden and increase the efficacy of immune therapy. Citation Format: Catherine B. MarElia-Bennett, Julia E. Lefler, Michael C. Ostrowski. A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-054.

Abstract PO-042: FGFR1 expression correlates inversely with the in vitro and in vivo efficacy of FGFR inhibitors in pancreatic cancer

Abstract Aberrant functions of fibroblast growth factor receptors (FGFR) have been implicated in the tumor progression of pancreatic cancer (PaCA). However, the mechanisms of FGFR action and the therapeutic utilities of FGFR inhibitors (FGFRi) in PaCA remain largely under-investigated. Here the mechanisms of FGFR inhibition in PaCA were investigated using pan-FGFRi that are under clinical development and specific shRNA against individual FGFRs. FGFRi treatment, such as BGJ398 and AZD4547, significantly inhibited cell proliferation, migration, and invasion, and cell expression of key markers associated with cell survival and invasiveness was suppressed, in multiple PaCA cell lines. Gemcitabine (Gem) is the standard-of-care for PaCA, and the development of Gem resistance (GemR) compromises its efficacy, thereby Gem only provides modest survival benefits. We found that FGFRi was effective in inhibiting cell proliferation, migration of strongly GemR cells and tumors (named as MIA-GR8 cells and MIA-GR8 tumors) that were all derived by increasing Gem exposure from MIA PaCa-2 cells in vitro and selected by Gem in vivo. These results suggest its potential clinical utility as a class of alternative drug candidates in tumors that acquired GemR. Downregulation of GemR markers mediated by FGFRi treatment in GemR cells also suggested its therapeutic application of FGFRi in combination with Gem, in order to overcome GemR. Mechanistically, the molecular mechanisms of individual FGFR were investigated. shRNA-mediated inhibition of FGFR2 or FGFR3 did not decrease cell growth, and the overexpression of FGFR1 promoted cell migration rather than proliferation. Furthermore, the efficacy of FGFRi treatment was investigated in multiple cell lines and patient-derived xenograft. The inhibitory effects of BGJ398 in PANC-1 cell growth and migration were compromised by exogenic overexpression of FGFR1, and its efficacy on cell viability suppression was correlated with FGFR1 in multiple cell lines. In patient-derived xenografts, the efficacy of FGFRi on tumor growth inhibition was also correlated with FGFR1 expression. These data provide evidence that precision medicine which targets FGFR using FGFRi in PaCA tumors which express relatively modest or low amount of FGFR1 could be more effective than that with a high FGFR1 expression. Citation Format: Qingxiang Lin, Andrea Serratore, Eugene Kandel, Robert M. Straubinger. FGFR1 expression correlates inversely with the in vitro and in vivo efficacy of FGFR inhibitors in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-042.

Diversity in the Extracellular Vesicle-Derived Microbiome of Tissues According to Tumor Progression in Pancreatic Cancer.

This study was conducted to identify the composition and diversity of the microbiome in tissues of pancreatic cancer and to determine its role. First, extracellular vesicles (EVs) were obtained from the paired tumor and normal tissues, and 16s rRNA gene sequencing was performed. We identified the microbiomes, compared the diversity between groups, and found that Tepidimonas was more abundant in tumors. Second, larger tumors resulted in lower levels of Leuconostoc and Sutterella, and increased lymph node metastasis resulted in higher levels of Comamonas and Turicibacter in tumor tissues. Moreover, in the case of tumor recurrence, the levels of Streptococcus and Akkermansia were decreased in tumor tissues. Finally, with the supernatant of Tepidimonas fonticaldi, proliferation and migration of cells increased, and epithelial-mesenchymal transition and the Tricarboxylic Acid (TCA) cycle-related metabolites were enhanced. The composition and diversity of EV-derived microbiomes are important for providing novel insights into theragnostic approaches in pancreatic cancer.

Hyperthermic Intra-Abdominal Chemotherapy with Gemcitabine as an Adjuvant Locoregional Treatment After Radical Surgery in Pancreatic Cancer

Purpose: The aim of our study is to present a series of patients in whom we have administered HIPEC therapy. Material and Methods: The treatment consists in performing cytoreductive surgery in patients diagnosed with potentially resectable pancreatic adenocarcinoma, together with the administration of HIPEC therapy with Gemcitabine. The dose of the drug is 120 mg/m2 for 30 minutes, followed by a conventional adjuvant treatment. Results: Five patients diagnosed with pancreatic adenocarcinoma have undergone surgery with a curative nature. There were no technical or hemodynamic complications during the procedure. Discussion: The use of HIPEC could achieve a decrease in the tumor progression of pancreatic cancer, improving the survival of the patient, and decreasing the recurrence of the disease. A larger number of patients will be necessary, as well as a long-term evaluation to assess the possible increase in survival in this group of patients.