Pancreatic Cancer Cell Survival Research Articles

Abstract C047: Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers

Abstract Oncogenic KRAS mutations present in 90–95% of pancreatic cancer patients. KRAS mutation in pancreatic cancer increased the dependency on glutamine and consumption of proline. Proline is a non-essential amino acid but it is important for the synthesis of proteins such as collagen, and also for precursors of antioxidant molecules. Several enzymes such as P5CS(ALDH18A1),PYCR1 and PYCR2 are involving proline synthesis. The effects of proline synthesis enzymes on pancreatic cancer cells are relatively less studied than that of enzymes catalyzing proline. Here, we examined the impact of proline synthesis enzymes on pancreatic cancer cell survival and proliferation. We first examined that exogenous proline promotes cell survival under low glutamine conditions in various pancreatic cancer cells. Exogenous proline supplementation increased some pancreatic cell survival even when glutamine was not sufficiently available, whereas proline supplement is dispensible for certain cell types of pancreatic cancer. Next, to investigate the importance of proline synthesis enzymes in cell survival, we measured the cell proliferation of pancreatic cancer with knock-down the proline synthesis enzymes. The knockdown of ALDH18A1 and PYCR1,2, which are proline biosynthesis enzyme genes, reduced cell survival in pancreatic cancer cells, suggesting that in PDAC cells, the enzymes that synthesize proline from glutamine are crucial for cancer cell survival and growth. To test the effect of proline-sysnthesis enzymes on autophagy, we measured autophagy flux in the cell harboring GFP-RFP-LC3 or GFP-LC3 as a reporter. Knockdown of proline synthesis enzymes, particularly ALDH18A1 or PYCR1 led to an increase in autophagy levels, indicating that proline-independent cells can bypass proline synthesis by autophagy induction in the absence of proline synthesis genes. Furthermore gemcitabine-resistant cells showed reduced levels of proline synthesis enzymes. In pancreatic cancer cell lines, proline promotes cancer cell survival, generated by both biosynthesis from glutamine and starvation-induced autophagy, which also critically important for supporting cellular drug resistance. Taken together, these results indicate that proline synthesis is mechanistically associated with autophagy induced by glutamine-starvation, which is pivotal for supplying proline in pancreatic cancer cells. Citation Format: Ji hyeon Kim, Heesun Cheong. Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C047.

Glutathione S-transferase-Pi 1 protects cells from irradiation-induced death by inhibiting ferroptosis in pancreatic cancer.

Glutathione S-transferase-Pi 1 (GSTP1) is an isozyme that plays a key role in detoxification and antioxidative damage. It also confers resistance to tumor therapy. However, the specific role of GSTP1 in radiotherapy resistance in pancreatic cancer (PC) is not known. In this study, we investigated how GSTP1 imparts radioresistance in PC. The findings of previous studies and this study revealed that ionizing radiation (IR) induces ferroptosis in pancreatic cancer cells, primarily by upregulating the expression of ACSL4. Our results showed that after IR, GSTP1 prolonged the survival of pancreatic cancer cells by inhibiting ferroptosis but did not affect apoptosis. The expression of GSTP1 reduced cellular ferroptosis by decreasing the levels of ACSL4 and increasing the GSH content. These changes increase the resistance of pancreatic cancer cells and xenograft tumors to IR. Our findings indicate that ferroptosis participates in irradiation-induced cell death and that GSTP1 prevents IR-induced death of pancreatic cancer cells by inhibiting ferroptosis.

Selective and effective suppression of pancreatic cancer through MNK inhibition

Objective The study aimed to explore the role of the Wnt/β-catenin signaling pathway in pancreatic cancer progression and chemoresistance, with a focus on identifying specific factors that distinguish between normal and tumor cells, thereby offering potential therapeutic targets. Materials and Methods We analyzed levels of total and phosphorylated eukaryotic translation initiation factor 4E (eIF4E) and β-catenin in pancreatic cancer and normal pancreatic tissues. Functional assays were used to assess the impact of eIF4E phosphorylation on β-catenin signaling, cell proliferation, and chemoresistance, with MNK kinase involvement determined through gene depletion studies. The MNK kinase inhibitor eFT508 was evaluated for its effects on eIF4E phosphorylation, β-catenin activation, and cell viability in both in vitro and in vivo models of pancreatic cancer. Results Both total and phosphorylated eIF4E, along with β-catenin, were significantly elevated in pancreatic cancer tissues compared to normal tissues. Phosphorylation of eIF4E at serine 209 was shown to activate β-catenin signaling, enhance cell proliferation, and contribute to chemoresistance in pancreatic cancer. Importantly, these effects were dependent on MNK kinase activity. Depletion of eIF4E reduced cell viability in both pancreatic cancer and normal cells, while depletion of MNK selectively decreased viability in pancreatic cancer cells. Treatment with eFT508 effectively inhibited eIF4E phosphorylation, suppressed β-catenin activation, and reduced pancreatic cancer cell growth and survival in vitro and in vivo, with minimal impact on normal cells. Conclusions: The MNK-eIF4E-β-catenin axis plays a critical role in pancreatic cancer progression and chemoresistance, distinguishing pancreatic cancer cells from normal cells. Targeting MNK kinases with inhibitors like eFT508 presents a promising therapeutic strategy for pancreatic cancer, with potential for selective efficacy and reduced toxicity.

Extracellular vesicles miR-31-5p promotes pancreatic cancer chemoresistance via regulating LATS2-Hippo pathway and promoting SPARC secretion from pancreatic stellate cells.

Pancreatic cancer remains one of the most lethal malignant diseases. Gemcitabine-based chemotherapy is still one of the first-line systemic treatments, but chemoresistance occurs in the majority of patients. Recently, accumulated evidence has demonstrated the role of the tumour microenvironment in promoting chemoresistance. In the tumour microenvironment, pancreatic stellate cells (PSCs) are among the main cellular components, and extracellular vesicles (EVs) are common mediators of cell‒cell communication. In this study, we showed that SP1-transcribed miR-31-5p not only targeted LATS2 in pancreatic cancer cells but also regulated the Hippo pathway in PSCs through EV transfer. Consequently, PSCs synthesized and secreted protein acidic and rich in cysteins (SPARC), which was preferentially expressed in stromal cells, stimulating Extracellular Signal regulated kinase (ERK) signalling in pancreatic cancer cells. Therefore, pancreatic cancer cell survival and chemoresistance were improved due to both the intrinsic Hippo pathway regulated by miR-31-5p and external SPARC-induced ERK signalling. In mouse models, miR-31-5p overexpression in pancreatic cancer cells promoted the chemoresistance of coinjected xenografts. In a tissue microarray, pancreatic cancer patients with higher miR-31-5p expression had shorter overall survival. Therefore, miR-31-5p regulates the Hippo pathway in multiple cell types within the tumour microenvironment via EVs, ultimately contributing to the chemoresistance of pancreatic cancer cells.

Retracted] Mammalian STE20‑like kinase 1 regulates pancreatic cancer cell survival and migration through Mfn2‑mediated mitophagy.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the JC‑1 staining images in Fig. 2C were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes that had either already been published elsewhere prior to the submission of this paper to Molecular Medicine Reports, or were under consideration for publication at around the same time (a small number of which have been retracted). In addition, the Snail western blot data in Fig. 3E bore a close similarity to certain of the Mfn2 data shown in Fig. 4A. In view of the fact that certain of the contentious data had already apparently been published previously, and owing to a lack of confidence in the presentation of certain of the data in this paper, the Editor of Molecular Medicine Reports has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 22: 398‑404, 2020; DOI: 10.3892/mmr.2020.11098].

The role of AMPK in pancreatic cancer: from carcinogenesis to treatment.

Pancreatic cancer has doubled over the previous two decades. Routine therapies are becoming incredibly resistant and failing to compensate for the burden caused by this aggressive neoplasm. As genetic susceptibility has always been a highlighted concern for this disease, identifying the molecular pathways involved in the survival and function of pancreatic cancer cells provides insight into its variant etiologies, one of which is the role of AMPK. This regulating factor of cell metabolism is crucial in the homeostasis and growth of the cell. Herein, we review the possible role of AMPK in pancreatic cancer while considering its leading effects on glycolysis and autophagy. Then, we assess the probable therapeutic agents that have resulted from the suggested pathways. Studying the underlying genetic changes in pancreatic cancer provides a chance to detect and treat patients suffering from advanced stages of the disease, and those who have given up their hope on conventional therapies can gain an opportunity to combat this cancer.

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.

OTUB1/NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

Pancreatic cancer is one of the most fatal cancers in the world. A growing number of studies have begun to demonstrate that mitochondria play a key role in tumorigenesis. Our previous study reveals that NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2), a core subunit of the mitochondrial respiratory chain complex I, is upregulated in Pancreatic adenocarcinoma (PAAD). However, its role in the development of PAAD remains unknown. Here, we showed that NDUFS2 played a critical role in the survival, proliferation and migration of pancreatic cancer cells by inhibiting mitochondrial cell death. Additionally, protein mass spectrometry indicated that the NDUFS2 was interacted with a deubiquitinase, OTUB1. Overexpression of OTUB1 increased NDUFS2 expression at the protein level, while knockdown of OTUB1 restored the effects in vitro. Accordingly, overexpression and knockdown of OTUB1 phenocopied those of NDUFS2 in pancreatic cancer cells, respectively. Mechanically, NDUFS2 was deubiquitinated by OTUB1 via K48-linked polyubiquitin chains, resulted in an elevated protein stability of NDUFS2. Moreover, the growth of OTUB1-overexpressed pancreatic cancer xenograft tumor was promoted in vivo, while the OTUB1-silenced pancreatic cancer xenograft tumor was inhibited in vivo. In conclusion, we revealed that OTUB1 increased the stability of NDUFS2 in PAAD by deubiquitylation and this axis plays a pivotal role in pancreatic cancer tumorigenesis and development.

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 392: MYB regulates a specific subset of hypoxia-induced genes and exhibits altered genomic occupancy under hypoxia

Abstract Hypoxia, an unavoidable microenvironmental stress, greatly influences the tumor cell behavior and therapeutic outcomes. Under low oxygen condition, tumor cells undergo transcriptional reprogramming, largely mediated through hypoxia-inducible factors (HIFs), triggering adaptive response pathways. We recently demonstrated a significant role of MYB in hypoxic survival of pancreatic cancer cells by facilitating adaptive changes in cellular metabolism in collaboration with HIF1α. Here, we employed genome-wide deep sequencing approaches to study the impact of hypoxia on transcriptional reprogramming of pancreatic cancer cells and investigate the role of MYB in the hypoxia-dependent processes. A large number of differentially expressed genes (DEGs) were identified in pancreatic cancer cells subjected to hypoxia (1% O2) and associated with multiple adaptive response pathways. Specifically, we found that pathways related to autophagy, cell migration, epithelial-to-mesenchymal transition, endocytosis, cytoskeleton reorganization were induced, while those associated with RNA processing, ribosome biogenesis, DNA replication and cell cycle were suppressed. To analyze the role of MYB in these adaptive responses, we compared the relative abundance of transcripts in MYB-overexpressing control and MYB knockout (KO) pancreatic cancer cells when cultured under normoxic or hypoxic conditions. We observed that the transcriptional output of MYB was drastically changed under hypoxia with a vast majority of genes being distinctly altered in response to the loss of MYB expression under normoxia and hypoxia. To examine if it resulted from altered genomic occupancy of MYB under normoxia and hypoxia, we compared MYB ChIP-seq peaks under these conditions. We observed a decrease in overall MYB-bound genomic regions under hypoxia despite the fact that we did not observe a decrease in nuclear MYB expression. More interestingly, the genomic occupancy of MYB shifted substantially from regions proximal to transcription start site to distal intergenic or intronic regions under hypoxia. Studies are currently underway to examine the mechanisms underlying the altered MYB genomic occupancy and its impact on adaptive physiological response of pancreatic cancer cells under hypoxia. Together, our findings bring unprecedented insight into the mechanistic role of MYB in hypoxia adaptation and pancreatic cancer pathobiology. Citation Format: Shashi Anand, Mohammad Aslam Khan, Kunwar Somesh Vikramdeo, Seema Singh, Ajay Pratap Singh. MYB regulates a specific subset of hypoxia-induced genes and exhibits altered genomic occupancy under hypoxia [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 392.

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 1796: Role of FASN in pancreatic neuroendocrine cancer cells survival

Abstract Introduction. Pancreatic neuroendocrine tumors (pNETs) are among the most frequently occurring neuroendocrine neoplasms. Up-regulation of fatty acid synthase (FASN) has been reported in many cancers, but the role of lipid metabolism in pNETs remains unclear. The purpose of this study was to determine whether FASN inhibition can be used as a target for pNET treatment. Methods. Immunohistochemistry and western blot analysis were used to determine protein levels of FASN in pNET tissues and cell lines. Expression of FASN was evaluated in pancreatic neuroendocrine primary and metastatic tumors and scored by a pathologist. Neutral lipid droplet levels in pNET cell lines were determined with BODIPY 493/503 staining. Clonogenic and SRB assays were used to evaluate cytotoxic effects of FASN inhibition by TVB-3664 in QGP-1 and BON pancreatic NET cell lines. Western blot analysis was used to determine levels of cleaved PARP and cyclin D1 after FASN inhibition. FASN knockdown was used to determine the effects of FASN downregulation on proliferation and lipid synthesis. Results. FASN expression was determined at the maximum scores of 6 and 5 in 98% of patient samples. BON, QGP-1 and NT-3 cell lines were stained with BODIPY 493/503; the highest levels of neutral lipid droplets were observed in the BON cell line. BON and QGP-1 cells were treated with TVB-3664 in either complete media with fatty acids (FA+) or complete media prepared with fatty acid depleted FBS (FA-). Treatment with TVB-3664 resulted in significant inhibition of BON and QGP-1 cell proliferation and cyclin D1 expression in both FA+ and FA- conditions. Depletion of extracellular FA enhanced the effect of TVB-3664 therapy on NET cells, especially in the BON cell line. FASN inhibition with TVB-3634 suppressed pNET cell proliferation and colony formation. Conclusions. Our study identified high protein levels of FASN in pNET patient samples and cell lines. Moreover, we demonstrated variability in the response of pNET cells to FASN inhibition. QGP cell viability was significantly decreased with FASN inhibition even in the presence of extracellular lipids. FASN inhibition in BON cells had only a minimal effect on cell viability and required depletion of extracellular lipids to achieve a decrease in cell viability and cyclin D1 expression. These data demonstrate that de novo lipid synthesis is involved in the pathogenesis and progression of pNETs. Importantly, our findings show that some pNETs are highly sensitive to FASN inhibition, suggesting that FASN inhibition may be considered as a treatment option in selected pNETs. Citation Format: Benjamin M. Evers, Zeta Chow, Yekaterina Zaytseva, Eun Y. Lee, Courtney M. Townsend, Tianyan Gao, B. Mark Evers, Piotr G. Rychahou. Role of FASN in pancreatic neuroendocrine cancer cells survival [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 1796.

Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival.

Pancreatic cancer is a leading cause of cancer-related deaths. Increased activity of the epidermal growth factor receptor (EGFR) is often observed in pancreatic cancer, and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration. Nevertheless, erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes. We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential (Δψm), facilitate tumor cell uptake of Δψm-sensitive agents, disrupt mitochondrial homeostasis, and subsequently trigger tumor cell death. Erlotinib has not been tested for this effect. To determine whether erlotinib can elevate Δψm and increase tumor cell uptake of Δψm-sensitive agents, subsequently triggering tumor cell death. Δψm-sensitive fluorescent dye was used to determine how erlotinib affects Δψm in pancreatic adenocarcinoma (PDAC) cell lines. The viability of conventional and patient-derived primary PDAC cell lines in 2D- and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone (MitoQ), a Δψm-sensitive MitoQ. The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0. The preclinical efficacy of the two-drug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts. Erlotinib elevated Δψm in PDAC cells, facilitating tumor cell uptake and mitochondrial enrichment of Δψm-sensitive agents. MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses, while erlotinib pretreatment potentiated low doses of MitoQ. SynergyFinder suggested that these drugs synergistically induced tumor cell lethality. Consistent with in vitro data, erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent. Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Repression of YEATS2 induces cellular senescence in hepatocellular carcinoma and inhibits tumor growth

ABSTRACT Hepatocellular carcinoma (HCC) stands as the third leading cause of cancer-related fatalities globally. In this study, we observed a significant increase in the expression level of the YEATS2 gene in HCC patients, and it is negatively correlated with the patients’ survival rate. While we have previously identified the association between YEATS2 and the survival of pancreatic cancer cells, the regulatory mechanisms and significance in HCC are still to be fully elucidated. Our study shows that knockdown (KD) of YEATS2 expression leads to DNA damage, which in turn results in an upregulation of γ-H2A.X expression and activation of the canonical senescence-related pathway p53/p21Cip1. Moreover, our transcriptomic analysis reveals that YEATS2 KD cells can enhance the expression of p21Cip1 via the c-Myc/miR-93-5p pathway, consequently fostering the senescence of HCC cells. The initiation of cellular senescence through dual-channel activation suggests that YEATS2 plays a pivotal regulatory role in the process of cell proliferation. Ultimately, our in vivo research utilizing a nude mouse tumor model revealed a notable decrease in both tumor volume and weight after the suppression of YEATS2 expression. This phenomenon is likely attributable to the attenuation of proliferative cell activity, coupled with a concurrent augmentation in the population of natural killer (NK) cells. In summary, our research results have supplemented the understanding of the regulatory mechanisms of HCC cell proliferation and indicated that targeting YEATS2 may potentially inhibit liver tumor growth.

Abstract C091: Harnessing proteostatic vulnerabilities in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) harbors mutations in KRAS in greater than 90% of cases and is characterized by a dense, fibrotic stroma that fosters the formation of a hypoxic, nutrient-poor tumor microenvironment, rendering tumor cells susceptible to proteotoxic stress. In order to prevent endoplasmic reticulum (ER) stress-induced cell death caused by accumulation of toxic protein aggregates, tumor cells must adapt proteostatic mechanisms including activation of the unfolded protein response (UPR) pathway and degradation of misfolded or aggregated proteins through proteasome-dependent or -independent mechanisms, such as ER-specific autophagy (ERphagy). Despite evidence suggesting that tumor cells may experience proteotoxic stress, little is known about the mechanisms pancreatic cancer cells employ to maintain proteostasis. We found that pancreatic cancer cells exhibit KRas-dependent constitutive UPR activation at the expense of ERphagy, suggesting that tumor cells preferentially employ UPR signaling to maintain proteostasis. Accordingly, mutant KRas pancreatic cancer cells have little to no aggregated proteins at baseline as a consequence of UPR activation. Furthermore, upon pharmacological enhancement of ER stress by 26S proteasome inhibition with bortezomib, ERphagy is engaged as a protective mechanism against UPR hyperactivation and apoptosis. In order to target protective ERphagy to promote apoptosis, we suppressed ERphagy activation through siRNA-mediated knockdown of ERphagy receptors under conditions of bortezomib-induced ER stress and found that depletion of CCPG1 but not other ERphagy receptors augmented the growth-suppressive effects of bortezomib due to greater UPR activation and accumulation of aggregated proteins. Overall, our findings implicate context-dependent reliance on both UPR signaling and ERphagy in pancreatic cancer cell survival and identify a targetable cell-intrinsic vulnerability that can leveraged for enhancing the cytotoxic effects of bortezomib, which alone is clinically ineffective in treating PDAC. Citation Format: Sandra Vogt, Dafna Bar-Sagi. Harnessing proteostatic vulnerabilities in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C091.

CA 19-9 but Not IGF-1/IGFBP-2 Is a Useful Biomarker for Pancreatic Ductal Adenocarcinoma (PDAC) and Chronic Pancreatitis (CP) Differentiation.

There are still no effective diagnostic and prognostic biomarkers in pancreatic ductal adenocarcinoma (PDAC). The differentiation between PDAC and chronic pancreatitis (CP) is often challenging. The inflammatory mass in the course of CP causes diagnostic difficulties in differentiating them from neoplastic lesions and, thus, delays the initiation of radical treatment. Insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 2 (IGFBP-2) form a network involved in PDAC development. The role of IGFs in promoting pancreatic cancer cell proliferation, survival, and migration is well established, and their ability to stimulate tumor growth and metastasis is well documented. The aim of the study was to evaluate the usability of IGF-1, IGFBP-2, and IGF-1/IGFBP-2 ratio in PDAC and CP differentiation. The study included 137 patients: 89 patients with PDAC and 48 patients with CP. All subjects were tested for the levels of IGF-1 and IGFBP-2 using the ELISA method (Corgenix UK Ltd. R&D Systems), along with the level of CA 19-9 in serum. Additionally, the IGF-1/IGFBP-2 ratio was calculated. Further analyses used logit and probit models with varying determinants in order to discern between PDAC and CP patients. The models served as a basis for AUROC calculation. The mean IGF-1 serum level was equal to 52.12 ± 33.13 ng/mL in PDAC vs. 74.23 ± 48.98 ng/mL in CP (p = 0.0053). The mean level of IGFBP-2 was equal to 305.95 ± 194.58 ng/mL in PDAC vs. 485.43 ± 299 ng/mL in CP (p = 0.0002). The mean CA 19-9 serum concentration was 434.95 ± 419.98 U/mL in PDAC vs. 78.07 ± 182.36 U/mL in CP (p = 0.0000). The mean IGF-1/IGFBP-2 ratio was 0.213 ± 0.14 in PDAC vs. 0.277 ± 0.33 in CP (p = 0.1914). The diagnostic usefulness of indicators for the purpose of PDAC and CP differentiation was assessed by means of AUROC comparison. The AUROCs of IGF-1, IGFBP-2, and IGF-1/IGFBP-2 ratio ranged below 0.7, being lower than the AUROC of CA 19-9 (0.7953; 0.719 within 95% CI). Together, the CA 19-9 and IGFBP-2 AUROCs also ranged below 0.8. When age was included, the AUROC increased to 0.8632, and its 95% confidence interval held above the 0.8 limit. The sensitivity of the used markers was not correlated to the stage of pancreatic PDAC. The presented results indicate that CA 19-9 is a marker demonstrating high potential for PDAC and CP differentiation. The inclusion of additional variables into the model, such as the serum level of IGF-1 or IGFBP-2, slightly increased the sensitivity in differentiating CP from PDAC. The IGF-1/IGFBP-2 ratio turned out to be a good marker of pancreatic diseases, but insufficient for the purpose of CP and PDAC differentiation.

USP33 enhances cell survival and stemness by deubiquitinating CTNNB1 in BXPC-3 and SW1990 cells.

Ubiquitin-specific protease 33 (USP33) has been implicated in various cancers, but its biological function and mechanism of action remain unknown in pancreatic cancer (PCa) as a deubiquitinating enzyme. Herein, we report that USP33 silencing inhibits PCa cell survival and self-renewal. USPs highly expressed in spherical PCa cells were screened by comparing the levels of ubiquitin-specific proteases in spherical PCa cells and adherent PCa cells. After silencing USP, the effect of USP on the proliferation of PCa cells was detected by CCK-8 and colony formation assay, and the effect of USP on cell stemness was detected by tumor sphere formation assay, flow analysis, and western blot analysis. The interaction of USP with CTNNB1 and the effect of USP on the ubiquitination of CTNNB1 were verified by coimmunoprecipitation assay. After replenishing CTNNB1, cell proliferation and cell stemness were examined. USP33 is upregulated in spheric BXPC-3, PCNA-1, and SW1990, compared with adherent BXPC-3, PCNA-1, and SW1990. USP33 interacts with CTNNB1, and stabilizes CTNNB1 by suppressing its degradation. Furthermore, cell proliferation, colony-forming, and self-renewal abilities of PCa cells in vitro, and the expression of stem cell markers EpCAM and CD44, C-myc, Nanog, and SOX2, were suppressed when USP33 was knocked down, which was reversed when CTNNB1 was ectopically expressed in PCa cells. Thus, USP33 promotes PCa cell proliferation and self-renewal by inhibiting the degradation of CTNNB1. USP33 inhibition may be a new treatment option for PCa patients.

Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine

BackgroundThe survival of pancreatic cancer cells, particularly cancer stem cells which are responsible for tumor relapse, depends on mitochondrial function. Mitochondrial transcription factor A (TFAM) is critical for the regulation of mitochondrial DNA and thus mitochondrial function. However, the possible involvement of TFAM in pancreatic cancer is unknown. MethodsHuman samples were obtained from pancreatic cancers and their adjacent tissues; human pancreatic cell lines were cultured in RPMI1640 medium. TFAM expressions in pancreatic tissues and cultured cells were determined using immunohistochemistry, ELISA, and reverse transcription polymerase chain reaction (RT-PCR). The effect of TFAM on cell growth, migration, colony formation and apoptosis were evaluated. Mitochondrial biogenesis in pancreatic cancer and normal cells were examined. ResultsThe majority of pancreatic cancer tissues exhibited higher TFAM expression compared to the adjacent counterparts. Consistently, TFAM mRNA and protein levels were higher in pancreatic cancer cell lines than in immortalized normal pancreatic epithelial cells. There was no difference on TFAM level between gemcitabine-sensitive and resistant pancreatic cancer cells. Functional analysis demonstrated that TFAM overexpression activated pancreatic normal and tumor cells whereas TFAM inhibition effectively inhibited the growth of pancreatic cancer cells. TFAM inhibition enhanced gemcitabine's cytotoxicity and suppressed growth, anchorage-independent colony formation and survival of gemcitabine-resistant pancreatic cancer cells. Mechanistic studies showed that TFAM inhibition resulted in remarkable mitochondrial dysfunction and energy crisis followed by oxidative stress. The basal mitochondrial biogenesis level correlated well with TFAM level in pancreatic cancer cells. ConclusionsTFAM played essential roles in pancreatic cancer via regulating mitochondrial functions which highlighted the therapeutic value of inhibiting TFAM to overcome gemcitabine resistance.

Mass Spectrometry Reveals High Levels of Hydrogen Peroxide in Pancreatic Cancer Cells

AbstractReactive oxygen species (ROS) are critical for many cellular functions, and dysregulation of ROS involves the development of multiple types of tumors, including pancreatic cancer. However, ROS have been grouped into a single biochemical entity for a long time, and the specific roles of certain types of ROS in tumor cells (e.g., pancreatic ductal adenocarcinoma (PDAC)) have not been systematically investigated. In this work, a highly sensitive and accurate mass spectrometry‐based method was applied to study PDAC cells of humans and of genetically modified animals. The results show that the oncogenic KRAS mutation promotes the accumulation of hydrogen peroxide (H2O2) rather than superoxide or hydroxyl radicals in pancreatic cancer cells. We further identified that the enriched H2O2 modifies cellular metabolites and promotes the survival of pancreatic cancer cells. These findings highlight the specific roles of H2O2 in pancreatic cancer development, which may provide new directions for pancreatic cancer therapy.

Mass Spectrometry Reveals High Levels of Hydrogen Peroxide in Pancreatic Cancer Cells.

Reactive oxygen species (ROS) are critical for many cellular functions, and dysregulation of ROS involves the development of multiple types of tumors, including pancreatic cancer. However, ROS have been grouped into a single biochemical entity for a long time, and the specific roles of certain types of ROS in tumor cells (e.g., pancreatic ductal adenocarcinoma (PDAC)) have not been systematically investigated. In this work, a highly sensitive and accurate mass spectrometry-based method was applied to study PDAC cells of humans and of genetically modified animals. The results show that the oncogenic KRAS mutation promotes the accumulation of hydrogen peroxide (H2 O2 ) rather than superoxide or hydroxyl radicals in pancreatic cancer cells. We further identified that the enriched H2 O2 modifies cellular metabolites and promotes the survival of pancreatic cancer cells. These findings highlight the specific roles of H2 O2 in pancreatic cancer development, which may provide new directions for pancreatic cancer therapy.