TOB1 suppresses proliferation in K-Ras wild-type pancreatic cancer.
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ABSTRACT:
SUBMITTER: Bai Y
PROVIDER: S-EPMC7013073 | biostudies-literature | 2020 Feb
REPOSITORIES: biostudies-literature
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Project description:Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.
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Project description:Tumor cells rely on aerobic glycolysis to generate ATP, namely the "Warburg" effect. 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, but its effect on cellular signaling pathways has not been fully elucidated. Herein, we sought to investigate the effect of 2-DG on ERK function in lung cancer cells. We found that 2-DG inhibits ERK phosphorylation in a time and dose-dependent manner in lung cancer cells. This inhibition requires functional LKB1. LKB1 knockdown in LKB1 wildtype cells correlated with an increase in the basal level of p-ERK. Restoration of LKB1 in LKB1-null cells significantly inhibits ERK activation. Blocking AMPK function with AMPK inhibitor, AMPK siRNA or DN-AMPK diminishes the inhibitory effect of 2-DG on ERK, suggesting that 2-DG-induced ERK inhibition is mediated by LKB1/AMPK signaling. Moreover, IGF1-induced ERK phosphorylation is significantly decreased by 2-DG. Conversely, a subset of oncogenic mutants of K-Ras, the main upstream regulator of ERK, blocks 2-DG-induced LKB1/AMPK signaling. These findings reveal the potential cross-talk between LKB1/AMPK and ERK signaling and help to better understand the mechanism of action of 2-DG.
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Project description:PD901, a MEK inhibitor, has been demonstrated of therapeutic efficacy against cholangiocarcinoma (CCA) harboring K-Ras oncogenic mutations. However, most CCA exhibit no K-Ras mutations. In the current study, we investigated the therapeutic potential of PD901, either alone or in combination with the pan-mTOR inhibitor MLN0128, for the treatment of K-Ras wild-type CCA in vitro using human CCA cell lines, and in vivo using AKT/YapS127A CCA mouse model. We discovered that in vitro, PD901 treatment strongly inhibited CCA cell proliferation, and combined PD901 and MLN0128 therapy further increased growth inhibition. In vivo, treatment of PD901 alone triggered tumor regression, which was not further increased when the two drugs were administered simultaneously. Mechanistically, PD901 efficiently hampered ERK activation in vitro and in vivo, leading to strong inhibition of CCA tumor cell cycle progression. Intriguingly, we discovered that PD901, but not MLN0128 treatment resulted in changes affecting the vasculature and cancer-associated fibroblasts in AKT/YapS127A mouse lesions. It led to the decreased hypoxia within tumor lesions, which may further enhance the anti-cell proliferation activities of PD901. Altogether, our study demonstrates that MEK inhibitors could be effective for the treatment of K-Ras wild-type CCA via inhibiting cell proliferation and modulating tumor microenvironment.
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Project description:Mesenchymal-epithelial transition factor (c-Met) is associated with the proliferation and motility of cancer cells. c-Met expression has been detected in surgical pancreatic cancer specimens, and its overexpression is associated with a poor prognosis. SU11274 is a specific inhibitor of c-Met. In the present study, the cell proliferation and motility of pancreatic cancer cells treated with SU11274 was investigated. The PANC-1, MIA-Paca2, NOR-P1, PK-45H, PK-1 and PK-59 pancreatic cancer cell lines were used. The expression of c-Met and cyclin D1 was analyzed by quantitative polymerase chain reaction. In addition, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess cell proliferation, and a scratch assay was performed to assess cell motility. c-Met expression was higher in PANC-1, PK-45H, PK-1 and PK-59 cell lines compared with that in normal pancreatic tissue. Following treatment with 30 µM SU11274, the proliferation of MIA-Paca2 and PK-45H cells was suppressed to 19.8±10.7% (P<0.05) and 45.8±14.8% (P<0.05) of the control level, respectively. Furthermore, cyclin D1 expression was downregulated to 43.7±17.9% (P<0.05) and 53.2±18.6% (P<0.05) of the control level in the MIA-Paca2 and PK-45H cell lines, respectively, following treatment with 30 µM SU11274. In addition, cell motility was reduced to 1.0±0.3% in MIA-Paca2 (P<0.05) and 14.7±3.5% in PK-45H (P<0.05) following treatment with 30 µM SU11274, compared with the motility of untreated cells. These results indicated that SU11274 suppresses the proliferation of pancreatic cancer cells via the downregulation of cyclin D1. The present study also demonstrated that cell motility was suppressed by treatment with SU11274.
| S-EPMC4533741 | biostudies-literature
Project description:Lessons learnedAntitumor activity was observed in the study population. Dose modifications of cabozantinib improve long-term tolerability. Biomarkers are needed to identify patient populations most likely to benefit. Further study of cabozantinib with or without panitumumab in patients with metastatic colorectal cancer is warranted.BackgroundThe epidermal growth factor receptor (EGFR) antibody panitumumab is active in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC), but nearly all patients experience resistance. MET amplification is a driver of panitumumab resistance. Cabozantinib is an inhibitor of multiple kinases, including vascular endothelial growth factor receptor 2 (VEGFR2) and c-MET, and may delay or reverse anti-EGFR resistance.MethodsIn this phase Ib clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of cabozantinib and panitumumab. We then treated an expansion cohort to further describe the tolerability and clinical activity of the RP2D. Eligibility included patients with KRAS WT mCRC (later amended to include only RAS WT mCRC) who had received prior treatment with a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab.ResultsTwenty-five patients were enrolled and treated. The MTD/RP2D was cabozantinib 60 mg p.o. daily and panitumumab 6 mg/kg I.V. every 2 weeks. The objective response rate (ORR) was 16%. Median progression free survival (PFS) was 3.7 months (90% confidence interval [CI], 2.3-7.1). Median overall survival (OS) was 12.1 months (90% CI, 7.5-14.3). Five patients (20%) discontinued treatment due to toxicity, and 18 patients (72%) required a dose reduction of cabozantinib.ConclusionThe combination of cabozantinib and panitumumab has activity. Dose reductions of cabozantinib improve tolerability.
| S-EPMC8176979 | biostudies-literature
Project description:Oncogenic, activating mutations in KRAS initiate pancreatic cancer. There are, however, two other Ras family members, Nras and Hras, which can be activated in the presence of oncogenic Kras. The role of these wild-type Ras proteins in cancer remains unclear, as their disruption has been shown to enhance or inhibit tumorigenesis depending upon the context. As pancreatic cancer is critically dependent upon Ras signaling, we tested and now report that loss of Hras increases tumor load and reduces survival in an oncogenic Kras-driven pancreatic adenocarcinoma mouse model. These effects were traced to the earliest stages of pancreatic cancer, suggesting that wild-type Hras may suppress tumor initiation. In normal cells, activated Ras can suppress proliferation through p53-dependent mechanisms. We find that the tumor suppressive effects of Hras are nullified in a homozygous mutant p53 background. As such, loss of wild-type Hras fosters the earliest stages of pancreatic cancer in a p53-dependent manner.
| S-EPMC4599940 | biostudies-literature
Project description:ObjectiveTo provide guidance for the management of RAS wild-type (wt) metastatic colorectal cancer (mCRC) in daily practice.MethodsNominal group and Delphi techniques were used. A steering committee of seven experts analyzed the current management of RAS wt mCRC, through which they identified controversies, critically analyzed the available evidence, and formulated several guiding statements for clinicians. Subsequently, a group of 30 experts (the expert panel) was selected to test agreement with the statements, through two Delphi rounds. The following response categories were established in both rounds: 1 = totally agree, 2 = basically agree, 3 = basically disagree, 4 = totally disagree. Agreement was defined if ≥ 75% of answers were in categories 1 and 2 (consensus with the agreement) or 3 and 4 (consensus with the disagreement).ResultsOverall, 71 statements were proposed, which incorporated the following areas: (1) overarching principles; (2) tumor location; (3) triplets; (4) maintenance; (5) second-line and beyond treatments; (6) Rechallenge and liquid biopsy. After the two Delphi rounds, only six statements maintained a lack of clear consensus.ConclusionsThis document aims to describe the expert's attitude when dealing with several common clinical questions regarding patients with RAS wt mCRC.
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Project description:The RAS family of oncogenes (HRAS, NRAS, and KRAS) are among the most frequently mutated protein families in cancers. RAS-mutated tumors were originally thought to proliferate independently of upstream signaling inputs, but we now know that non-mutated wild-type (WT) RAS proteins play an important role in modulating downstream effector signaling and driving therapeutic resistance in RAS-mutated cancers. This modulation is complex as different WT RAS family members have opposing functions. The protein product of the WT RAS allele of the same isoform as mutated RAS is often tumor-suppressive and lost during tumor progression. In contrast, RTK-dependent activation of the WT RAS proteins from the two non-mutated WT RAS family members is tumor-promoting. Further, rebound activation of RTK-WT RAS signaling underlies therapeutic resistance to targeted therapeutics in RAS-mutated cancers. The contributions of WT RAS to proliferation and transformation in RAS-mutated cancer cells places renewed interest in upstream signaling molecules, including the phosphatase/adaptor SHP2 and the RasGEFs SOS1 and SOS2, as potential therapeutic targets in RAS-mutated cancers.
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Project description:Pancreatic cancer is a deadly disease that is frequently associated with mortality at the time of diagnosis due to rapid metastasis, which makes it unsuitable for operative surgery, and resistant to chemotherapy and radiation therapy. Isochorismatase domain-containing protein 1 (ISOC1) has putative isochorismatase activity, and is positively regulated by estrogen in human breast cancer. However, its role in pancreatic cancer has yet to be fully elucidated. Analysis from datasets downloaded from The Cancer Genome Atlas and Genotype-Tissue Expression databases indicated that the ISOC1 mRNA expression level was increased in pancreatic cancer tissues, compared with normal pancreatic tissues. In the present study, it was determined that the human pancreatic cancer cell lines SW 1990, PANC-1 and AsPC-1 had increased expression levels of ISOC1 mRNA, compared with human pancreatic ductal epithelial cells. Additionally, two of the pancreatic cancer cell lines, SW 1990 and PANC-1, transfected with lentivirus-delivered short hairpin RNA, to knockdown the expression of ISOC1, were established. Cell counting and MTT assays indicated that knockdown of ISOC1 decreased the ability of cell growth and proliferation in pancreatic cancer cells. Furthermore, Annexin V staining and caspase-3/7 activity assays demonstrated that inhibition of ISOC1 promoted cell apoptosis via elevation of the expression of caspase-3/7. Furthermore, inhibition of ISOC1 impaired the cell migration and invasive capability of the cells. In conclusion, ISOC1 exerts a role in pancreatic cancer cell growth and apoptosis, and may have a role in pancreatic cancer tumorigenesis.
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Project description:Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional ?-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.
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