Radioresistance Of Pancreatic Cancer Cells Research Articles

MiR-6855-5p Enhances Radioresistance and Promotes Migration of Pancreatic Cancer by Inducing Epithelial-Mesenchymal Transition via Suppressing FOXA1: Potential of Plasma Exosomal miR-6855-5p as an Indicator of Radiosensitivity in Patients with Pancreatic Cancer.

Whether radiation should be added to neoadjuvant treatment remains controversial, and liquid biopsy has not been reported to predict radioresistance in pancreatic cancer (PC). We aimed to identify microRNAs (miRNAs) governing radioresistance in PC by utilizing peripheral plasma exosome samples and to verify their usefulness as biomarkers. miRNA microarray analysis was conducted using pretreatment peripheral plasma exosomes from 10 patients with PC receiving neoadjuvant chemoradiotherapy (NACRT) in the discovery cohort. Patients were categorized into two groups (good and poor responders) based on treatment responses, and candidate miRNAs exhibiting differential expression between the two groups were identified. The radiosensitivity of PC cells was examined after miR-6855-5p overexpression. Next-generation sequencing (NGS) and TargetScan were used to explore the mechanisms of radioresistance. We investigated the correlation between miR-6855-5p expression levels in the pretreatment peripheral plasma exosomes of 28 patients in the validation cohort and the response to NACRT. miR-6855-5p expression was higher in poor responders than in good responders. miR-6855-5p induces radioresistance in PC cells. NGS showed that epithelial-mesenchymal transition (EMT) was involved in miR-6855-5p-related radioresistance. Forkhead box protein A1 (FOXA1) was identified as a direct target of miR-6855-5p using NGS and TargetScan. Clinical examination of samples from the validation cohort revealed a tendency for patients with higher expression of miR-6855-5p in peripheral plasma exosomes to exhibit increased radioresistance (r=-0.5964). miR-6855-5p regulates the radioresistance of PC by inducing EMT via suppressing FOXA1, and miR-6855-5p in peripheral plasma exosomes may be a biomarker for radioresistance of PC.

MiR-193a-5p Enhances the Radioresistance of Pancreatic Cancer Cells by Targeting ZFP57 and Activating the Wnt Pathway.

This study was to investigate whether miR-193a-5p and ZFP57 are involved in the radioresistance of pancreatic cancer and to explore its working mechanism. Pancreatic cancer tissues were harvested from patients who achieved CR (complete remission) and PR (partial remission) and those who achieved PD (progressive disease) and SD (stable disease). The mRNA and protein expressions of ZFP57 and miR-193a-5p were determined by RT-qPCR and WB (Western blot), respectively. For in vitro experiments, the parental BxPC-3 cell line was irradiated by X-ray at a total dose of 40 Gy to induce the irradiation-resistant subtype BxPC-3-RR. ZFP57 was downregulated in radioresistant pancreatic cancer cells. The results of dual-luciferase reporter gene assay, RNA pull-down assay, RT-qPCR, and WB confirmed that miR-193a-5p targeted ZFP57 and inhibited ZFP57 expression. The MTT assay and the colony formation assay showed that the radioresistant pancreatic cancer cells had higher viability and survival fraction. The results of WB indicated that in the radioresistant pancreatic cancer cells, the cyclin D1, Bax, CDk4, cleaved caspase-3, Bcl-2, and γ-H2AX proteins were upregulated to varying degrees. The results of the in vitro nude mouse experiment were consistent with those of in vivo experiments. According to the cell transfection and salvage experiments, miR-193a-5p down regulated ZFP57 after radiotherapy. As a result, the Wnt pathway was activated, which further induced radioresistance of pancreatic cancer cells. Our experiments showed that the miR-193a-5p/ZFP57/Wnt pathway mediated the radioresistance of pancreatic cancer cells, providing novel clues for the treatment of pancreatic cancer.

Upregulation of CD73 Confers Acquired Radioresistance and is Required for Maintaining Irradiation-selected Pancreatic Cancer Cells in a Mesenchymal State

The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics analysis of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor-/cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT, and MAPK/ERK pathways, were activated in the radioresistant cells, leading to inhibition of apoptosis and increased epithelial-mesenchymal plasticity. In addition, the radioresistant cells exhibited enhanced capabilities of DNA repair and antioxidant defense compared with the parental cells. We focused functional analysis on one of the most up-regulated proteins in the radioresistant cells, ecto-5'-nucleotidase (CD73), which is a cell surface protein that is overexpressed in different types of cancer. Ectopic overexpression of CD73 in the parental cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 re-sensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies demonstrate that CD73 up-regulation promotes Ser-136 phosphorylation of the proapoptotic protein BAD and is required for maintaining the radioresistant cells in a mesenchymal state. Our findings suggest that expression alterations in the IR-selected pancreatic cancer cells result in hyperactivation of the growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity and enhancement of DNA repair. Our results also suggest that CD73, potentially a novel downstream factor of the enhanced growth factor/cytokine signaling, confers acquired radioresistance by inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136 and by maintaining the radioresistant pancreatic cancer cells in a mesenchymal state.

EP-2165 m6A RNA modification by METTL3 regulates chemo-and radioresistance in pancreatic cancer cells

EP-2165 m6A RNA modification by METTL3 regulates chemo-and radioresistance in pancreatic cancer cells

Adaptive EGF expression sensitizes pancreatic cancer cells to ionizing radiation through activation of the cyclin D1/P53/PARP pathway.

It is well-known that the activation status of the P53, signal transducer and activator of transcription(Stat)3 and nuclear factor (NF)‑κB signaling pathways determines the radiosensitivity of cancer cells. However, the function of these pathways in radiosensitive vs radioresistant cancer cells remains elusive. The present study demonstrated that adaptive expression of epidermal growth factor (EGF) following exposure to ionizing radiation (IR) may induce radiosensitization of pancreatic cancer(PC) cells through induction of the cyclinD1/P53/poly(ADP‑ribose) polymerase pathway. By contrast, adaptively expressed interleukin(IL)‑6 and insulin‑like growth factor(IGF)‑1 may promote radioresistance of PC cells, likely through activation of the Stat3 and NF‑κB pathways. In addition, cyclinD1 and survivin, which are specifically expressed in the G1/S and G2/Mphase of the cell cycle, respectively, are mutually exclusive in radiosensitive and radioresistant PC cells, while Bcl‑2 and Bcl‑xL expression does not differ between radiosensitive and radioresistant PC cells. Therefore, adaptively expressed EGF and IL‑6/IGF‑1 may alter these pathways to promote the radiosensitivity of PC cancers. The findings of the present study highlight potential makers for the evaluation of radiosensitivity and enable the development of effective regimens for cancer radiotherapy.

MiR-590-5p Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Radiation by Blocking Autophagy via Targeting ATG3

Background: Resistance against radio therapy is growing concern in treating patients of pancreatic cancer (PC). Improving our depth with regards to mechanisms involved in radio resistance could help in developing strategies for increasing patient's response to this therapy. Here we investigated the role of micro RNAs (miRs) and specifically miR-590-5p in radio-resistance of PC cells. Methods: We successfully developed radio resistant PC cell lines in our lab and subjected them to microarray analysis to compare the levels of miRs with their original parental cell lines. We then transfected the PC cells using miR mimics or inhibitor and performed clonogenic survival assay to study the effects on radiation sensitivity. We also studied the effect of miR-590-5p on autophagy using electron microscopy and Immunoblotting analysis. Luciferase assay was done for identifying mRNA targets of miR-590-5p. Results: The radio resistant PC cells exhibited decreased expression of miR-590-5p with elevated autophagy against the parental cells. The over expression of miR-590-5p inhibited the radiation mediated autophagy and the miR-590-5p inhibitor encouraged autophagy in PC cells. The up-regulation of miR-590-5p enhanced the sensitivity of PC cells towards radiation. We confirmed that ATG-3 as a favorable target of miR-590-5p and was unregulated in radio resistant cells, we also found that ATG-3 was associated with autophagy. MiR-590-5p inhibited radiation-mediated autophagy and enhanced radio sensitivity of PC cells. The results suggested an inverse correlation for miR-590-5p and autophagy in PC tissues. Conclusions: The expression of miR-590-5p in PC cells is associated with elevated levels of autophagy along with ATG-3, which leads to radio resistance; miR-590-5p can be employed to enhance the radio sensitivity of PC cells. Funding Statement: The project was self financed. Declaration of Interests: The authors have no conflict of interest. Ethics Approval Statement: All the patients were educated about the study and informed consent forms were signed before the study. The study was approved from institutional review of research committee at The Second Xiangya Hosipital of Central South University, Changsha, Hunan, China

Prolyl hydroxylase domain 3 influences the radiotherapy efficacy of pancreatic cancer cells by targeting hypoxia-inducible factor-1α.

PurposePancreatic cancer is characterized by a hypoxic microenvironment and resistance to most currently available treatment modalities. Prolyl hydroxylase domain 3 (PHD3) is a rate-limiting enzyme that regulates the degradation of hypoxia-inducible factors (HIFs) and is deregulated in pancreatic cancer cells. Whether such alteration of PHD3 expression contributes to the sustained growth and radioresistance of pancreatic cancer cells remains largely unknown.Materials and methodsPHD3 was overexpressed in pancreatic cancer Mia-paca2 cells via lentiviral expression. Cell cycle progression and apoptosis were assayed by flow cytometry. HIF-1α, EGFR, and PHD3 protein expression was assessed by Western blotting. Cell survival was determined in a colony formation assay.ResultsPHD3 overexpression suppressed HIF-1α protein expression and EGFR phosphorylation and enhanced the 2 Gy irradiation-mediated reductions in HIF-1α and phosphorylated (p)-EGFR under either normoxic or hypoxic conditions. PHD3 overexpression inhibited the growth and colony formation of Mia-paca2 cells in response to irradiation under either normoxic or hypoxic conditions. PHD3 overexpression exacerbated irradiation-induced apoptosis, with a greater effect under hypoxia than normoxia. Cell cycle distribution analysis demonstrated that PHD3 overexpression resulted in further shortened S phase and lengthened G2/M phase in response to irradiation.ConclusionPHD3 expression may contribute to the radiotherapy efficacy of pancreatic cancer cells and serve as a novel biomarker for improving radiotherapy efficacy in pancreatic cancer.

The relationship between the radioresistance of pancreatic cancer cell SW1990 and the induction of the Epithelial-Mesenchymal Transition: an in vitro study

Objective: To investigate the relationship between the radioresistance of pancreatic cancer cell SW1990 and the induction of the Epithelial-Mesenchymal Transition (EMT). Methods: The radio-resistant pancreatic cancer cell SW1990-R were established by using the method of radiating pancreatic cancer cell SW1990 step by step and repeatedly. Then the changes of the morphology of the cell was observed by inverted phase contrast microscope, the radioresistance of SW1990-R was detected by colony-forming assay, and the apoptosis rate of the two cell lines after radiation were measured by flow cytometry. Then invasiveness and EMT-related genes was measured by trans-well test and qRT-PCR. Finally, the model of transplanted tumor on nude mouse was used to confirm the relationship between the radioresistance of pancreatic cancer cell SW1990 and the induction of EMT. Results: Compared with SW1990, SW1990-R had a lower radiosensitivity (survival fraction in 2 Gy, SF2: 0.326 3±0.007 3 vs 0.840 8±0.001 9, P<0.05) and lower apoptosis rate[(6.12±1.27) % vs (16.87±1.73)%, P<0.05]. Meanwhile, the invasive ability of SW1990-R were significant higher than that of SW1990 cell. According to the result of both in vivo and in vitro experiment, SW1990-R had a higher expression level of Vimentin and Snail, and lower expression level of E-cadherin when compared with SW1990. Conclusion: Compared with SW1990, the radio-resistant pancreatic cancer cell SW1990-R can induce the Epithelial-mesenchymal transition.

Inhibition of PI3K/AKT Signaling Pathway Radiosensitizes Pancreatic Cancer Cells with ARID1A Deficiency in Vitro.

Pancreatic cancer is among the most aggressive human cancers, and is resistant to regular chemotherapy and radiotherapy. The AT-rich interactive domain containing protein 1A (ARID1A) gene, a crucial chromatin remodeling gene, mutates frequently in a broad spectrum of cancers, including pancreatic cancer. Recent evidence suggests that ARID1A acts as tumor suppressor and plays an important role in DNA damage repair (DDR). However, the effect of ARID1A on the radiosensitivity of pancreatic cancer remains unclear. Herein, we investigated the involvement of ARID1A depletion in the radioresistance of pancreatic cancer cells, and explored the underlying mechanisms. The results reveal that knockdown of ARID1A enhances the radioresistance of pancreatic cancer cells through suppressing apoptosis, impairing G2-M checkpoint arrest, strengthening DDR, and accompanying activation of PI3K/AKT signaling pathway. Moreover, upon inhibition of PI3K/AKT pathway by PI3K-inhibitor LY294002 or AKT-inhibitor mk2206, the radiosensitivity of ARID1A-deficient pancreatic cancer cells is improved in vitro via increased apoptosis and weakened DDR. Taken together, these data suggest that loss of ARID1A expression enhances radioresistance of pancreatic cancer through activation of PI3K/AKT pathway, which maybe a promising target for radiosensitization of ARID1A-deficient pancreatic cancer.

Nanoparticle Augmented Radiotherapy using Titanium Oxide Nanoparticles

Titanium oxide is a photoactive material that generates hydroxyl free radicals via water splitting. When doped with rare earth ions titanium oxide nanoparticles are activated by X-rays and X-ray generated electrons and are used to enhance radiotherapy treatment of solid tumours. As the nanoparticles generate free radicals by water splitting the presence of molecular oxygen is not required and aggressive hypoxic tumours may be targeted. A clonogenic assay of radio resistant pancreatic cancer (PANC- 1) cells shows a radiotherapy dose enhancement factor of 1.9 at clinically relevant nanoparticle loadings. A fast growing oropharyngeal cancer (FaDu) xenograft demonstrates that rare earth doped titanium oxide nanoparticles delivered by intratumoural injection disperse throughout the tumour, being taken up by cancer cells and undergoing passive accumulation in the Golgi apparatus. Incident radiotherapy activates the nanoparticles to produce hydroxyl free radicals, destroying the Golgi apparatus, and inducing tumour cell apoptosis. This results in a reduction in proliferating cancer cells and a consequent reduction in tumour regrowth rate by a factor of 3.8. There is no increase in systemic toxicity when using nanoparticles in addition to radiotherapy. Rare earth doped titanium oxide nanoparticles therefore represent a novel approach to tumour treatment via destruction of the cells Golgi apparatus during radiotherapy.

The epitranscriptome m6A writer METTL3 promotes chemo- and radioresistance in pancreatic cancer cells.

N6-methyladenosine (m6A) is the most abundant epitranscriptome modification in mammalian mRNA. Recent years have seen substantial progress in m6A epitranscriptomics, indicating its crucial roles in the initiation and progression of cancer through regulation of RNA stabilities, mRNA splicing, microRNA processing and mRNA translation. However, by what means m6A is dynamically regulated or written by enzymatic components represented by methyltransferase-like 3 (METTL3) and how m6A is significant for each of the numerous genes remain unclear. We focused on METTL3 in pancreatic cancer, the prognosis of which is not satisfactory despite the development of multidisciplinary therapies. We established METTL3-knockdown pancreatic cancer cell line using short hairpin RNA. Although morphologic and proliferative changes were unaffected, METTL3-depleted cells showed higher sensitivity to anticancer reagents such as gemcitabine, 5-fluorouracil, cisplatin and irradiation. Our data suggest that METTL3 is a potent target for enhancing therapeutic efficacy in patients with pancreatic cancer. In addition, we performed cDNA expression analysis followed by gene ontology and protein-protein interaction analysis using the Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins databases, respectively. The results demonstrate that METTL3 was associated with mitogen-activated protein kinase cascades, ubiquitin-dependent process and RNA splicing and regulation of cellular process, suggesting functional roles and targets of METTL3.

The CD44 standard isoform contributes to radioresistance of pancreatic cancer cells.

Resistance to chemoradiotherapy is one reason for the increased recurrence rate of pancreatic cancer after these therapies. These cells change the expression levels of several proteins, such as epithelial–mesenchymal transition (EMT), while acquiring the chemo- or radio-resistance. In this study, we focused on CD44, a pancreatic cancer stem cell marker. CD44 has isoforms with different functions: standard isoform (CD44s) and several variant isoforms (CD44v). However, little is known about the roles of these isoforms after ionizing irradiation. The purpose of this study was to investigate the role of CD44 isoforms in radioresistance of pancreatic cancer cells. AsPC-1 (a human pancreatic cancer cell line) was irradiated with 4 MV X-rays. The mRNA and protein levels of CD44s were strongly upregulated, dose dependently, compared with CD44v after irradiation. Thus, we further investigated CD44s at the point of cell proliferation. We evaluated cell proliferation and survival, using CD44s knockdown cells. CD44s knockdown did not change the proliferation rate for up to 72 h after the irradiation, but decreased cell viability in the colony formation assay. As one of the reasons for these effects, we found downregulation of phosphorylated extracellular signal–regulated kinase (Erk; which is involved with cell proliferation) by CD44s knockdown, time dependently. Moreover, radiation-induced EMT-like expression changes were detected and suppressed by CD44s knockdown. In conclusion, our work demonstrated that CD44 standard isoform was especially upregulated after high-dose X-ray irradiation in several isoforms of CD44 and contributed to longer-term cell survival after the irradiation through the maintenance of Erk phosphorylation and radiation-induced EMT.

Abstract B05: Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer

Abstract Pancreatic cancer is an exceptional aggressive cancer, and acquired radiation resistance in this type of cancer is common. The objective of this study was to use a comparative proteomic method to identify the proteins that are responsible for acquired radioresistance in pancreatic cancer cells. In order to eliminate the complications caused by genetic background variations, we first generated a pancreatic cancer isogenic stable cell line that differs in radioresistance. The isogenic stable cell line was generated by exposing the pancreatic cancer MIA PaCa-2 cells (an undifferentiated pancreatic cancer cell line) to a total dose of 60 Gy of ionizing radiation (IR; gamma radiation) in 30 fractions over a period of 6 weeks, a protocol that is commonly used in clinical radiotherapy. The clones that survived from the fractionated exposures showed strong radioresistance, which could be demonstrated by both cell apoptotic assays and the long-term clonogenic survival assays. We then used a mass spectrometry-based quantitative proteomic method to systematically compare the expression of proteins between the radiosensitive parental cells and the selected radioresistant cells. One protein identified to be substantially upregulated in the radioresistant cells was ecto-5’-nucleotidase (CD73), a cell surface enzyme that converts adenosine monophosphate (AMP) into adenosine and is known to play important roles in cell-cell/cell-matrix interactions and immune suppression. To examine whether the upregulated CD73 was functionally related to the acquired radioresistance of pancreatic cancer cells, we silenced or ectopically overexpressed CD73 gene in the cells and examined the effect of the expression manipulations on long-term survival and apoptosis of the cells in response to IR exposure. The results demonstrated that knockdown of CD73 in the radioresistant cells significantly decreased the long-term survival rates, and resensitized the cells to apoptosis after the cells were exposed to IR. Concomitantly, stable overexpression of CD73 in the radiosensitive parental cells significantly increased the long-term survival rates, and enhanced resistance of the cells to apoptosis upon exposure of the cells to IR. To determine if the enzyme activity of CD73 is important for its effect on radioresistance, we generated an enzyme-dead mutant CD73 and overexpressed the mutant CD73 in the parental cells. Functional assays demonstrated that the mutant CD73 affected radioresistance of pancreatic cancer cells in a similar way as wild-type CD73, suggesting that the enzyme activity is not related to its effect on radioresistance. In summary, our results suggest that upregulation of CD73 expression is an important mechanism by which pancreatic cancer cells acquire resistance to IR, and the effect of CD73 on radioresistance in pancreatic cancer cells is enzyme-independent and at least partially through affecting the sensitivity of the cells to IR-induced apoptosis. Citation Format: Anna M. Nguyen, Jianhong Zhou, Yuchun Du.{Authors}. Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B05.

Abstract 1669: Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation

Abstract Radiation therapy is a staple treatment for pancreatic cancers. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both G1/S and G2/M checkpoints. However, cancer cells are often defective in G1/S checkpoint due to mutations/alterations in the key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras mediated transformation. Here, we show that Rac1 and its downstream effector, the PAK kinases, also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1/PAK pathway with pharmacological inhibitors (Ehop-016, AZA1, NSC23766, and FRAX597) or a dominant negative mutant of Rac1 (Rac1T17N) blocks activation of the ATM/Chk2 and ATR/Chk1 pathways, abrogates activation of the IR-induced G2/M checkpoint, delays the repair of IR-induced double-stranded DNA breaks, and increases the radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer. Citation Format: Michel M. Ouellette, Surinder K. Batra, Ying Yan. Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1669.

MicroRNA-216a enhances the radiosensitivity of pancreatic cancer cells by inhibiting beclin-1-mediated autophagy

Radioresistance has become a challenge in the treatment of pancreatic cancer, which limits the efficacy and outcomes of radiotherapy in clinical treatment. Autophagy, recognized as an adaptive response to cell stress, has recently been involved in the radioresistance of cancer cells. MicroRNAs (miRNAs) are also involved in the radioresistance of pancreatic cancer cells. In the present study, we established a radioresistant pancreatic cancer cell line and found that miRNA-216a was significantly downregulated whereas the autophagy activity was increased as compared with the control. Forced expression of miR-216a was found to inhibit the expression of beclin-1, a critical autophagic gene, as well as autophagy. Using bioinformatics analysis and the dual-luciferase reporter gene assay, we found that miR-216a directly interacted with 3'-untranslated region (UTR) of beclin-1. Furthermore, the forced expression of miR‑216a inhibited cell growth and colony formation ability and promoted the cell apoptosis of radioresistant pancreatic cancer cells in response to irradiation. By contrast, overexpression of beclin-1 abrogated the effects of miR-216a. Furthermore, miR-216a sensitized xenograft tumor to irradiation treatment and inhibited irradiation-induced autophagy by regulating beclin-1. Collectively, the results demonstrated that miR‑216a enhanced the radiosensitivity of pancreatic cancer cells by inhibiting beclin-1-mediated autophagy, suggesting a promising molecular target for improving the radiotherapy of pancreatic cancer.

LIM-only protein FHL2 critically determines survival and radioresistance of pancreatic cancer cells

Numerous factors determine the current poor prognosis of pancreatic ductal adenocarcinoma (PDAC). One of the greatest challenges to overcome is treatment resistance. Among a large repertoire of intrinsic resistance mechanisms, integrin-mediated cell adhesion to extracellular matrix (ECM) has been identified to be fundamental. Coalesced in focal adhesion complexes, integrins, receptor tyrosine kinases, protein kinases and adapter proteins mediate prosurvival signaling. Four and a half LIM domains protein 2 (FHL2) is one of these adapter proteins, which operates through protein–protein interactions and shows tumor-specific expression. Based on this, we investigated FHL2 expression in PDAC specimens and three-dimensionally grown cell lines and how FHL2 mechanistically contributes to cell survival, cell cycling and radiation resistance. PDAC exhibited a significantly increased and heterogeneous FHL2 expression. Upon FHL2 depletion, pancreatic cancer cell lines showed significantly decreased cell survival, proliferation and radioresistance as well as enhanced apoptosis and MEK/ERK signaling and cyclin D1, E, A and B1 expression were strongly induced. Targeting of FHL2 and MEK1 was similarly effective than FHL2 depletion alone, suggesting MEK1 as a downstream signaling mediator of FHL2. Taken together, our results provide evidence for the importance of the focal adhesion protein FHL2 in pancreatic cancer cell survival, proliferation and radiosensitivity.

Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

Radiation therapy is a staple treatment for pancreatic cancer. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both a G1 and G2 checkpoint. However, cancer cells are often defective in G1 checkpoint due to mutations/alterations in key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras-mediated transformation. Here, we show that Rac1 also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1 using specific inhibitor and dominant negative Rac1 mutant not only abrogates IR-induced G2 checkpoint activation, but also increases radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer.

Contextual regulation of pancreatic cancer stem cell phenotype and radioresistance by pancreatic stellate cells

Background and purposeProgression of pancreatic ductal adenocarcinoma (PDAC) is promoted by desmoplasia induced by pancreatic stellate cells (PSC). Contributory to this progression is epithelial mesenchymal transition (EMT), which shares many characteristics with the cancer stem cell (CSC) hypothesis. We investigated the role of these processes on the radioresponse and tumorigenicity of pancreatic cancer cells. Materials and methodsWe used an in vitro sphere model and in vivo xenograft model to examine the role of PSC in EMT and CSC processes. ResultsWe demonstrated that PSC enhanced the CSC phenotype and radioresistance of pancreatic cancer cells. Furthermore, the expression of several EMT and CSC markers supported enhanced processes in our models and that translated into remarkable in vivo tumorigenicity. Multi-dose TGFβ neutralizing antibody inhibited the EMT and CSC processes, sensitized cells to radiation and reduced in vivo tumorigenicity. A proteomic screen identified multiple novel factors that were regulated by PSC in pancreatic cells. ConclusionThese results are critical in highlighting the role of PSC in tumor progression and radioresistance by manipulating the EMT and CSC processes. TGFβ and the novel factors identified are important targets for better therapeutic outcome in response to PSC mediated mechanisms.

ATDC/TRIM29 Phosphorylation by ATM/MAPKAP Kinase 2 Mediates Radioresistance in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by therapeutic resistance for which the basis is poorly understood. Here, we report that the DNA and p53-binding protein ATDC/TRIM29, which is highly expressed in PDAC, plays a critical role in DNA damage signaling and radioresistance in pancreatic cancer cells. Ataxia-telangiectasia group D-associated gene (ATDC) mediated resistance to ionizing radiation in vitro and in vivo in mouse xenograft assays. ATDC was phosphorylated directly by MAPKAP kinase 2 (MK2) at Ser550 in an ATM-dependent manner. Phosphorylation at Ser-550 by MK2 was required for the radioprotective function of ATDC. Our results identify a DNA repair pathway leading from MK2 and ATM to ATDC, suggesting its candidacy as a therapeutic target to radiosensitize PDAC and improve the efficacy of DNA-damaging treatment.

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