Growth Of Pancreatic Cancer Cells Research Articles

TAGLN2 targeted control of ARPC5-mediated activation of the MEK/ERK signaling pathway influences the proliferation, invasion, and metastasis of pancreatic cancer cells

PurposePancreatic cancer (PC) is a common tumor of the digestive tract with an insidious onset and high malignancy potential. Currently, surgery is the only effective treatment modality. Therefore, it is crucial to discover new targeted therapeutic modalities. We studied whether transgelin 2 (TAGLN2) targeted control of actin-related protein 2/3 complex subunit 5 (ARPC5)-mediated activation of the MEK/ERK signaling pathway to Influences the proliferation, invasion, and metastasis of pancreatic cancer cells. MethodsThe effects of TAGLN2 overexpression and knockdown on the proliferative viability and invasive metastatic ability of pancreatic cancer cells were verified through in vitro and in vivo assays via constructing a stable lentiviral transfection of human pancreatic cancer cell lines PANC-1 and SW1990. Bioinformatics analysis was used to predict the relationship between TAGLN2 and ARPC5. These findings were subsequently verified through protein profiling, immunofluorescence (IF), and coimmunoprecipitation (CO-IP) assays. In vitro experiments were also conducted to confirm the effect of TAGLN2 modulation on ARPC5 expression, which subsequently affects the proliferation and invasive metastatic ability of pancreatic cancer cells. The study analyzed the relationship between TAGLN2 and the MEK/ERK signaling pathway through bioinformatics and in vitro experiments with the MEK signaling pathway inhibitor U0126. ResultsTAGLN2 is expressed at high levels in pancreatic cancer cell lines, and its expression is positively correlated with poor prognosis of pancreatic cancer. ARPC5 is a direct target of TAGLN2 and is associated with the MEK/ERK signaling pathway. In vivo and ex vivo experiments confirmed that overexpression of TAGLN2 promoted the proliferation, invasion, and metastasis of pancreatic cancer cells, and silencing ARPC5 reversed these effect. ConclusionOur research revealed that TAGLN2 protein binds to ARPC5 protein and contributes to increased ARPC5 expression and activation of the MEK/ERK signaling pathway. This activation promotes pancreatic cancer cell growth, infiltration, and spread. Hence, TAGLN2 is a potential viable therapeutic target in pancreatic cancer and represents a novel therapeutic approach.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Design, synthesis and evaluation of a pyrazolo[3,4-d]pyrimidine derivative as a novel and potent TGFβ1R1 inhibitor

The transforming growth factor β1 (TGFβ1)/SMAD signaling pathway regulates many vital physiological processes. The development of potent inhibitors targeting activin receptor-like kinase 5 (ALK5) would provide potential treatment reagents for various diseases. A significant number of ALK5 inhibitors have been discovered, and they are currently undergoing clinical evaluation at various stages. However, the clinical demands were far from being met. In this study, we utilized an alternative conformation-similarity-based virtual screening (CSVS) combined with a fragment-based drug designing (FBDD) strategy to efficiently discover a potent and active hit with a novel chemical scaffold. After structural optimization in the principle of group replacement, compound 57 was identified as the most promising ALK5 inhibitor. Compound 57 demonstrated significant inhibitory effects against the TGF-β1/SMAD signaling pathway. It could markedly attenuate the production of extracellular matrix (ECM) and deposition of collagen. Also, the lead compound showed adequate pharmacokinetic (PK) properties and good in vivo tolerance. Moreover, treatment with compound 57 in two different xerograph models showed significant inhibitory effects on the growth of pancreatic cancer cells. These results suggested that lead compound 57 refers as a promising ALK5 inhibitor both in vitro and in vivo, which merits further validation.

The UBE2F-CRL5ASB11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells

UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Abstract 4128: Biofield therapy suppressed invasion and metastases of human pancreatic cancer

Abstract The NIH categorizes Biofield Therapy (BT) as therapeutic approaches within energy medicine that involve using the body's energy field (biofield) for therapeutic benefit. Although controversial, in some cases devices have been developed that mimic the electromagnetic fields (EMF) that are emitted from people when delivering BTs. We previously reported that BT significantly inhibited the growth of pancreatic cancer cells and liver metastasis in their relevant animal models mediated in part through modification of the cell cycle, reductions in cell voltage potentials, and down regulation of PI3K/Akt pathways. We expanded this research to examine the potential role of BT in anti-invasiveness in human pancreatic ductal adenocarcinoma (PDAC) cells and animal models. We found that PANC-1 cells treated with BT (15 min) displayed 55% less invading cells than that of untreated incubator control and sham control cells (P < 0.001). Similar results were observed in human PDAC L3.7 cells and mouse pancreatic cancer KPCY cells. These experiments were replicated 8 times for Panc-1 cells and 4 times for L3.7 cells. Additionally, migration capacity was tested using the transwell migration and scratch assays. Consistent with the result of cell invasion assays, BT treatment also led to significant reduction of migration of PANC-1 and L3.7 cells by 34% and 41%, respectively, compared to sham controls. Two additional therapists independently replicated the anti-invasiveness and anti-migratory effects of BT on these PDAC cells. This suggests the replicability of these findings across studies and therapists. In a repeated PANC-1 mouse orthotopic model, we found that only a third of BT treated mice had visible liver nodules whereas liver metastasis was observed in more than half of mice in the untreated colony control and sham control groups. Furthermore, BT treatment significantly reduced liver tumor burden by 73% compared to that of the sham control group (p < 0.05), which was similar to that observed in the previous study. Similar to the prior PANC-1 mouse model, in the current study BT treatment had only a moderate impact on the growth of primary pancreatic tumor. We assessed certain markers indicative of cell status as well as pathways associated with migration and invasion and these will be presented at the meeting. Our findings suggest that exposure to BT reduced cellular invasion and migration processes and profoundly reduced metastasis of pancreatic tumor in an animal model. This study is in part supported by Emerald Gate Charitable Trust. Citation Format: Peiying Yang, Sharmistha Chakraborty, Phuong Nguyen, Defeng Deng, Andrew Cusimano, Daoyan Wei, Lorenzo Cohen. Biofield therapy suppressed invasion and metastases of human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4128.

Abstract 1591: Hepatocytes compensate for the loss of PHGDH in pancreatic cancer cells during liver metastasis

Abstract Background: Pancreatic cancer (PC), one of the deadliest malignancies, exhibits a high rate of distant metastasis that directly correlates with the mortality rate. Liver serves as one of the most predominant sites of metastasis in PC patients. The nutrient availabilities at the metastatic organ and the metabolic dependencies of the invading tumor cells play pivotal role in shaping the metastatic tumor microenvironment (TME) and establishing metastatic colonies. We observed an elevated expression of PHGDH, the first rate-limiting enzyme of the serine (Ser) biosynthesis pathway, in the hepatocytes (most abundant liver-resident cells) isolated from murine pancreatic liver-metastasis (LM), as compared to normal livers. Hence, we hypothesized that hepatocytes may promote the growth of PC cells by sufficing for their Ser-requirement during LM. Methods: Murine PC line (HY19636 derived from LSL-KrasG12D; p53 L/+, Ptf1a-Cre+ tumors), were used to generate LMs in B6 mice via hemi-splenic route. PHGDH was knocked-out by lentiviral and AAV-mediated delivery of CRISPR-Cas9 gRNAs in vitro and in vivo, respectively. RNA-seq, immunoblot, immunohistochemistry, confocal microscopy, ELISA, flow cytometry, chromatin immunoprecipitation and promoter-reporter analyses were performed to identify the molecular axis, followed by functional validations using genetic and pharmacologic inhibitions in murine and human lines. Frozen homogenates with matched formalin-fixed sections of LMs from PC patients provided a unique correlative platform for the study. Results: A significant portion of the LM tissues (68% in our cohort of 32 PC patients) demonstrated no to very low expression of PHGDH in the cancer cells, rendering them as exogeneous Ser (exSer)-dependent. Notably, loss of PHGDH in the tumor cells induced an elevated PI3K/AKT signaling, downstream of a CXCL5/CXCR2 axis, in the neighboring hepatocytes. This led to FOXO3A’s cytoplasmic sequestration and decline in its occupancy on PHGDH promoter, causing a transcriptional upregulation of PHGDH in these hepatocytes, which enabled them to supply Ser and support the growth of exSer-dependent cancer cells in the liver. Abrogation of each node in the CXCR2-PI3K-FOXO3A axis and hepatocyte-specific deletion of PHGDH, led to a significant decline in the metastatic burden of exSer-dependent cells in the liver with a significant increase in overall mice survival, particularly under Ser-deprived dietary conditions. Conclusion: Our findings demonstrate a novel cancer cell-hepatocytes crosstalk necessary for the growth of PC cells in Ser-depleted conditions. Here, we have identified several actionable signaling nodes in the liver that serve as compensatory metabolic allies for PC cells during liver metastasis, and can be considered as potential therapeutic targets in adjuvant setting. Citation Format: Koelina Ganguly, Keisuke Yamamoto, Joel Encarnacion Rosado, Albert Sohn, Douglas Biancur, Elaine Lin, Elshadai White, Alec C. Kimmelman. Hepatocytes compensate for the loss of PHGDH in pancreatic cancer cells during liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1591.

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7628.

Abstract 4391: The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells

Abstract UBE2F is a neddylation E2 that couples with RBX2/SAG E3 to promote CUL-5 neddylation and Cullin-RING ligase-5 (CRL5) activation. Whether and how UBE2F regulates pancreatic tumorigenesis is previously unknown. Here we showed that UBE2F is growth essential for pancreatic cancer cells with KRAS mutant in culture. In KrasG12D mouse PDAC model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signal pathways via blocking ubiquitylation and degradation of Diras2, which is a new substrate of Cul5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of pancreatic cancer cells with KRAS mutant, and Diras2 deletion largely rescued the phenotypes induced by Ube2f deletion, indicating its causal role. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in KrasG12D PDAC model, respectively. The Ube2f-CRL5Asb11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients. Citation Format: Yi Sun, Yu Chang, Qian Che, Hua Li, Jie Xu, Mingjia Tan, Xiufang Xiong. The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4391.

Abstract 5742: Therapeutic efficacy of CA102N in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer to treat due to its aggressive nature and late-stage diagnosis. Considering the lack of effective therapies, there is an urgent need to develop new and effective therapeutics to improve the prognosis of patients. CA102N is a carrier-mediated novel anticancer product where hyaluronic acid is conjugated to Nimesulide-NH2. CA102N is currently under development for the treatment of solid tumors, including colorectal cancer. CA102N was safe and well tolerated in the first-in-human trial in patients with locally advanced or metastatic solid tumors. In the present study, the in vitro anticancer activity of CA102N was investigated in K-ras mutant PANC-1 pancreatic cancer cells. The antitumor efficacy was also tested in vivo in the PANC-1 mice model. Results demonstrated that CA102N inhibits cell growth, cell migration, angiogenesis, and cell proliferation in both K-ras mutant PANC-1 pancreatic cancer cells or tumors. Administration of CA102N in PANC-1 xenografts resulted in a dose-dependent and statistically significant decrease in tumor growth at 9.8 mg/kg and 14.7 mg/kg Nim equivalents, respectively, compared to the vehicle control group. A significant decrease (80%) in liver metastasis was also noted in the PANC-1 orthotopic mice model at 19.6 mg/kg Nim equivalents dose level. Mechanistically, CA102N was found to suppress the PI3k/Akt/mTOR and Raf/MEK/ERK signaling pathways, in addition to reducing the expressions of the N6-methyladenosine (m6A) reader and writer proteins, OCT3/4 transcription factor and RAS levels in vivo. CA102N also modified the tumor stroma and improved drug permeability in vivo. Based on this compelling data, CA102N shows promise as a potential treatment for pancreatic cancer. Combination therapies and regimens are currently being explored in in vivo models as a strategy for clinical development. Citation Format: Louis Lin, Shuling Tu, Eskouhie Tchaparian. Therapeutic efficacy of CA102N in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5742.

Abstract 3050: Evaluating the role of ∆Np63α in driving growth and metastasis of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the US and is rising in incidence. PDAC tumors may be either classical or basal in transcriptomic subtype, and PDAC patients with basal tumors have worse prognosis and may have increased resistance to chemotherapy. Previous studies have shown that ∆Np63α drives basal program expression. The goal of this project is to investigate whether ∆Np63α expression is sufficient to increase pancreatic cancer cell growth, metastasis and chemoresistance and whether these cells can outcompete ∆Np63α non-expressers in mixed tumors. Non-∆Np63α expressing human PDAC cell lines AsPC1 and HPAFII were stably transduced to express either ∆Np63α or mCherry (control). For both AsPC1 and HPAFII, ∆Np63α-expressing and control cells had similar proliferation rates and susceptibility to chemotherapy agents oxaliplatin and SN38 when grown in culture. When control or ∆Np63α-expressing AsPC1 cells were implanted orthotopically into the pancreas of athymic nude mice, there was no significant difference in primary tumor mass and pathological analysis of lungs and livers did not reveal any significant difference in metastatic burden. Bulk RNA sequencing of primary tumors confirmed that tumors in mice inoculated with AsPC1-∆Np63α had significantly upregulated expression of genes associated with the basal subtype. Collectively, these data suggest that while ∆Np63α drives the basal program in PDAC, its expression is insufficient to cause resistance to these chemotherapies or a growth or metastatic advantage in an immunodeficient setting. Further work is needed to elucidate the mechanism by which ∆Np63α contributes to more aggressive PDAC tumors. Citation Format: Daniel H. Ryan, Chin-Hsien (Emily) Tai, Xianyu Zhang, Mayrel Palestino Dominguez, Christine Alewine. Evaluating the role of ∆Np63α in driving growth and metastasis of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3050.

The Effect of 4-(Dimethylamino)phenyl-5-oxopyrrolidines on Breast and Pancreatic Cancer Cell Colony Formation, Migration, and Growth of Tumor Spheroids.

A series of hydrazones, azoles, and azines bearing a 4-dimethylaminophenyl-5-oxopyrrolidine scaffold was synthesized. Their cytotoxic effect against human pancreatic carcinoma Panc-1 and triple-negative breast cancer MDA-MB-231 cell lines was established by MTT assay. Pyrrolidinone derivatives 3c and 3d, with incorporated 5-chloro and 5-methylbenzimidazole fragments; hydrazone 5k bearing a 5-nitrothien-2-yl substitution; and hydrazone 5l with a naphth-1-yl fragment in the structure significantly decreased the viability of both cancer cell lines. Compounds 3c and 5k showed the highest selectivity, especially against the MDA-MB-231 cancer cell line. The EC50 values of the most active compound 5k against the MDA-MB231 cell line was 7.3 ± 0.4 μM, which were slightly higher against the Panc-1 cell line (10.2 ± 2.6 μM). Four selected pyrrolidone derivatives showed relatively high activity in a clonogenic assay. Compound 5k was the most active in both cell cultures, and it completely disturbed MDA-MB-231 cell colony growth at 1 and 2 μM and showed a strong effect on Panc-1 cell colony formation, especially at 2 μM. The compounds did not show an inhibitory effect on cell line migration by the 'wound-healing' assay. Compound 3d most efficiently inhibited the growth of Panc-1 spheroids and reduced cell viability in MDA-MB-231 spheroids. Considering these different activities in biological assays, the selected pyrrolidinone derivatives could be further tested to better understand the structure-activity relationship and their mechanism of action.

TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor growth and metastasis. The current therapies do not include strategies against pro-tumorigenic inflammation in cancer patients. We have shown that the upregulated cell surface expression of Toll-like Receptor (TLR) 2 and of TLR9 inside PDAC cells maintain chronic inflammatory responses, support chemotherapeutic resistance, and mediate tumor progression in human pancreatic cancer. We further demonstrated intracellular TLR2 and TLR9 targeting using specific intrabodies, which resulted in downregulated inflammatory signaling. In this study, we tested, for the first time, an intrabody-mediated TLR blockade in human TLR2- and TLR9-expressing pancreatic cancer cells for its effects on inflammatory signaling-mediated tumor growth. Newly designed anti-TLR2- and anti-TLR9-specific intrabodies inhibited PDAC growth. Co-expression analysis of the intrabodies and corresponding human TLRs showed efficient retention and accumulation of both intrabodies within the endoplasmic reticulum (ER), while co-immunoprecipitation studies indicated both intrabodies interacting with their cognate TLR antigen within the pancreatic cancer cells. Cancer cells with attenuated proliferation expressing accumulated TLR2 and TRL9 intrabodies demonstrated reduced STAT3 phosphorylation signaling, while apoptotic markers Caspases 3 and 8 were upregulated. To conclude, our results demonstrate the TLR2 and TLR9-specific intrabody-mediated signaling pathway inhibition of autoregulatory inflammation inside cancer cells and their proliferation, resulting in the suppression of pancreatic tumor cell growth. These findings underscore the potential of specific intrabody-mediated TLR inhibition in the ER relevant for tumor growth inhibition and open up a new therapeutic intervention strategy for the treatment of pancreatic cancer.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

DT-13 inhibits the proliferation of pancreatic cancer by inducing apoptosis via AMPK-mTOR signaling

Background/ObjectiveDT-13, the principal active component of Mysidium shortscapes from the Liliaceae family, has garnered substantial interest in cancer therapy owing to its potential anticancer properties. This study investigated the effects of DT-13 on the proliferation and apoptosis of human pancreatic cancer cell lines and aimed to elucidate the underlying mechanisms. MethodsPANC1 and CFPAC1 cells were exposed to DT-13 and their proliferation was assessed using RTCA and clone formation assays. Apoptotic protein expression was analyzed by western blotting, and apoptotic cells were identified by flow cytometry. RNA was extracted from DT-13 treated and untreated PANC1 cells for RNA sequencing. Differentially expressed genes were identified and subjected to GO bioprocess, KEGG pathway analysis, and western blotting. Finally, to evaluate tumor growth, CFPAC1 cells were subcutaneously injected into BALB/c nude mice. ResultsDT-13 inhibited proliferation and induced apoptosis of PANC1 and CFPAC1 cells by activating the AMPK/mTOR pathway and suppressing p70 S6K. Moreover, DT-13 hindered the growth of CFPAC1 xenograft tumors in nude mice. ConclusionsDT-13 effectively inhibited the growth of human pancreatic cancer cells.

Discovery of Novel PDEδ Autophagic Degraders: A Case Study of Autophagy-Tethering Compound (ATTEC).

The autophagy-tethering compound (ATTEC) technology has emerged as a promising strategy for targeted protein degradation (TPD). Here, we report the discovery of the first generation of PDEδ autophagic degraders using an ATTEC approach. The most promising compound 12c exhibited potent PDEδ binding affinity and efficiently induced PDEδ degradation in a concentration-dependent manner. Mechanistic studies confirmed that compound 12c reduced the PDEδ protein level through lysosome-mediated autophagy without affecting the PDEδ mRNA expression. Importantly, compound 12c was much more effective in suppressing the growth in KRAS mutant pancreatic cancer cells than the corresponding PDEδ inhibitor. Taken together, this study expands the application scope of the ATTEC approach and highlights the effectiveness of the PDEδ autophagic degradation strategy in antitumor drug discovery.

Functional structural domain synthesis of anti-pancreatic carcinoma pectin-like polysaccharide RN1

The great structural and functional diversity supports polysaccharides as favorable candidates for new drug development. Previously we reported that a drug candidate pectin-like natural polysaccharide, RN1 might target galectin-3 (Gal-3) to impede pancreatic cancer cell growth in vivo. However, the quality control of polysaccharide-based drug research faces great challenges due to the heterogeneity. A potential solution is to synthesize structurally identified subfragments of this polysaccharide as alternatives. In this work, we took RN1 as an example, and synthesized five subfragments derived from the putative repeating units of RN1. Among them, pentasaccharide 4 showed an approximative binding affinity to Gal-3 in vitro, as well as an antiproliferative activity against pancreatic BxPC-3 cells comparable to that of RN1. Further, we scaled up pentasaccharide 4 to gram-scale in an efficient synthetic route with a 6.9 % yield from D-galactose. Importantly, pentasaccharide 4 significantly suppressed the growth of pancreatic tumor in vivo. Based on the mechanism complementarity of galactin-3 inhibitor and docetaxel, the combination administration of pentasaccharide 4 and docetaxel afforded better result. The result suggested pentasaccharide 4 was one of the functional structural domains of polysaccharide RN1 and might be a leading compound for anti-pancreatic cancer new drug development.

Abstract LB_C13: Development of BLX-3030, a potent, selective, orally available CDK9i shows promise in Pancreatic Ductal Adenocarcinoma (PDAC) models

Abstract Background: CDK9 (cyclin-dependent kinase 9), is a Ser/Thr kinase that plays a crucial role in regulating gene expression. It is a component of the P-TEFb complex, which controls transcription elongation by phosphorylating RNA polymerase II. Dysregulated CDK9 has been linked to cancer progression. Pharmacological CDK9 inhibition has recently emerged as a novel and promising approach for cancer therapy, particularly in exocrine pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (PNETs). PDAC and PNETs are cancer entities with a dismal prognosis, owing mainly to their resistance to chemo, radiation, and immunotherapies. Dysregulation of CDK9 activity can lead to uncontrolled cell cycle progression and tumor formation. Given the role of CDK9 in promoting PDACs progression, targeting this kinase has emerged as a potential therapeutic strategy for PDACs and PNETs where our late-breaking results of BLX-3030 demonstrated efficacy in pre-clinical tumor models. Materials and Methods: Our proprietary MolecuLernTM fragment-based design platform, in vitro cellular efficacies as a single agent or combination, FACS/apoptosis, cell migration, live-cell imaging, and immunofluorescence assays. Top compounds were evaluated for 7-day tox, PK, selectivity, Nano BRET, ADME-Tox, in vitro safety, hERG, P450, SafetyScreen44 panel, LDH-Glo assay, and KINOMEscan for selectivity. Results: Utilizing a CDK9-refined crystal structure and the MolecuLernTM, we identified novel, reversible, ATP-competitive inhibitors that exhibited potent activity in MYC-expressing pancreatic cancer cells in vitro. BLX-3030, elected from over 90 analogs demonstrated potent activity with an IC50 of 4.2 nM in inhibiting CDK9. CDKs family selectivity and target engagement NanoBRET assay results show the BLX-3030 selectivity and its on-target potency with an IC50 of 24.3 nM. BLX-3030 potently inhibited the growth of pancreatic cancer cells with IC50s ranging from 133 nM to 508 nM in 10 different cell lines. Furthermore, BLX-3030 and its analogs showed excellent PK properties in mice, rats, and dogs with an oral bioavailability of 28%, 38%, and 83%, respectively. BLX-3030 has a low clearance and >3 hours half-life in rodents and canine species. The effect of BLX-3030 in vivo PDAC models including ex vivo results for the levels of CDK9, pCDK9, c-MYC, and cleaved Caspase 3 in tumor tissues will be discussed. Based on these pre-clinical results, BLX-3030 was nominated as a candidate with additional efficacy and safety data supporting IND-enabling studies Conclusion: In summary, BLX-3030 is a potent CDK9i that displayed potent antitumor activity in pancreatic cancer models, with its promising safety, PK, and developable characteristics leading us to select and nominate as clinical candidates. Our late-breaking pre-clinical results from our in vivo study in pancreatic cancer tumor models, including 7-day safety, tolerability, and toxicokineCDK9tic results received are supportive for our planned ND enabling studies and future clinical trials will be presented. Citation Format: Kyle Medley, Zhaoliang Li, Chenyu Lin, Yesenia Barrera-Millan, Lynne Kelley, Haiyong Han, Hariprasad Vankayalapati, David J Bearss. Development of BLX-3030, a potent, selective, orally available CDK9i shows promise in Pancreatic Ductal Adenocarcinoma (PDAC) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C13.

Phytochemicals for the prevention and treatment of pancreatic cancer: Current progress and future prospects.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States, owing to its aggressive nature and suboptimal treatment options, emphasizing the need for novel therapeutic approaches. Emerging studies have exhibited promising results regarding the therapeutic utility of plant-derived compounds (phytochemicals) in pancreatic cancer. The purpose of this review is to evaluate the potential of phytochemicals in the treatment and prevention of pancreatic cancer. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses was applied to collect articles for this review. Scholarly databases, including PubMed, Scopus and ScienceDirect, were queried for relevant studies using the following keywords: phytochemicals, phenolics, terpenoids, alkaloids, sulfur-containing compounds, in vitro, in vivo, clinical studies, pancreatic cancer, tumour, treatment and prevention. Aggregate results pooled from qualified studies indicate phytochemicals can inhibit pancreatic cancer cell growth or decrease tumour size and volume in animal models. These effects have been attributed to various mechanisms, such as increasing proapoptotic factors, decreasing antiapoptotic factors, or inducing cell death and cell cycle arrest. Notable signalling pathways modulated by phytochemicals include the rat sarcoma/mitogen activated protein kinase, wingless-related integration site/β-catenin and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signal transduction pathways. Clinically, phytochemicals have been found to increase survival while being well-tolerated and safe, though research is scarce. While these promising results have produced great interest in this field, further in-depth studies are required to characterize the anticancer activities of phytochemicals before they can be utilized to prevent or treat pancreatic cancer in clinical practice.

A benzochalcone derivative synchronously induces apoptosis and ferroptosis in pancreatic cancer cells

Background Pancreatic cancer is a highly aggressive and lethal disease with limited treatment options. In this study, we investigated the potential therapeutic effects of compound KL-6 on pancreatic cancer cells. Methods The study involved assessing the inhibitory effects of KL-6 on cell proliferation, clonogenic potential, cell cycle progression, apoptosis, migration, and invasion. Additionally, we examined the action mechanism of KL-6 by RNA-seq and bioinformatic analysis and validated by qRT-PCR and western blot in pancreatic cancer cells. Results Our results demonstrated that KL-6 effectively inhibited the growth of pancreatic cancer cells in a dose-dependent manner. It induced G2/M phase cell cycle arrest and apoptosis, disrupting the cell cycle progression and promoting cell death. KL-6 also exhibited inhibitory effects on cell migration and invasion, suggesting its potential to suppress the metastatic properties of pancreatic cancer cells. Furthermore, KL-6 modulated the expression of genes involved in various cancer-related pathways including apoptosis and ferroptosis. Conclusion These findings collectively support the potential of KL-6 as a promising therapeutic option for pancreatic cancer treatment. Further research is needed to fully understand the underlying mechanisms and evaluate the clinical efficacy of KL-6 in pancreatic cancer patients.

A prognostic and immunotherapy effectiveness model for pancreatic adenocarcinoma based on cuproptosis-related lncRNAs signature.

Pancreatic adenocarcinoma (PAAD) results in one of the deadliest solid tumors with discouraging clinical outcomes. Growing evidence suggests that long non-coding RNAs (lncRNAs) play a crucial role in altering the growth, prognosis, migration, and invasion of pancreatic cancer cells. Cuproptosis is a novel type of cell death induced by copper (Cu) and is associated with mitochondrial respiration during the tricarboxylic acid cycle. However, the relationship between lncRNAs related to cuproptosis and PAAD is poorly studied. In this study, we investigated the association between a signature of cuproptosis-related lncRNAs and the diagnosis of PAAD. Genomic data and clinical information were obtained using the TCGA dataset, while cuproptosis-related genes (CRGs) from previous studies. Co-expression analysis was utilized to identify lncRNAs associated with cuproptosis. We developed and verified a prognostic risk model following a classification of patients into high- and low-risk categories. The prediction capacity of the risk model was assessed using a number of methods including Kaplan-Meier analysis, receiver operating characteristic (ROC) curves, nomograms, and principal component analysis (PCA). Furthermore, differentially expressed genes (DEGs) were used to perform functional enrichment analyses, and to examine the behaviors of various risk groups in terms of immune-related activities and medication sensitivity. We identified 7 cuproptosis-related lncRNA signatures, including CASC19, FAM83A-AS1, AC074099.1, AC007292.2, AC026462.3, AL358944.1, and AC009019.1, as overall survival (OS) predictors. OS and progression-free survival (PFS) showed significant differences among patients in different risk groups. Independent prognostic analysis revealed that the cuproptosis-related lncRNA signatures can independently achieve patient prognosis. The risk model demonstrated strong predictive ability for patient outcomes, as evidenced by ROC curves, nomograms, and PCA. Higher tumor mutation burden (TMB) and lower tumor immune dysfunction and exclusion (TIDE) scores were observed in the high-risk group. Additionally, the low-risk group was hypersensitive to 3 anti-cancer medications, whereas the high-risk group was hypersensitive to one. A prognostic risk model with a good predictive ability based on cuproptosis-related lncRNAs was developed, providing a theoretical basis for personalized treatment and immunotherapeutic responses in pancreatic cancer.