Our previous study showed that Calreticulin (CRT) promoted the development of pancreatic cancer (PC) through ERK/MAPK pathway. We next investigate whether CRT promotes EGF-induced epithelial–mesenchymal transition (EMT) in PC via Integrin/EGFR-ERK/MAPK signaling, which has not been reported yet to our knowledge. EGF simultaneously induced EMT and activated Integrin/EGFR–ERK/MAPK signaling pathway in 3 PC cells. However, CRT silencing significantly inhibited EGF function, including inhibiting EGF-induced EMT-like cell morphology, EGF-enhanced cell invasion and migration, and EGF induced the decrease of E-cadherin, ZO-1, and β-catenin and the increase of the key proteins in Integrin/EGFR-ERK/MAPK signaling (pEGFR-tyr1173, Fibronectin, Integrinβ1, c-Myc and pERK). Conversely, CRT overexpression rescued the change of EMT-related proteins induced by EGF in CRT silencing PC cells. Additionally, CRT was co-stained with pEGFR1173 (with EGF), Fibronectin and Integrinβ1 by IF under confocal microscopy and was co-immunoprecipitated with Fibronectin, Integrinβ1 and c-Myc in both PC cells, all of which indicating a close interaction of CRT with Integrin/EGFR–ERK/MAPK signaling pathway in PC. In vivo, CRT silencing inhibited subcutaneous tumor growth and liver metastasis of pancreatic tumor. A positive relationship of CRT with Fibronectin, Integrinβ1, c-Myc and pERK and a negative association of CRT with E-cad was also observed in vivo and clinical samples. Meanwhile, overexpression of the above proteins was closely associated with multiple aggressive clinicopathological characteristics and the poor prognosis of PC patients. CRT promotes EGF-induced EMT in PC cells via Integrin/EGFR-ERK/MAPK signaling pathway, which would be a promising therapy target for PC.
We studied the clinicopathological significance for Calreticulin (CRT) expression in pancreatic cancer (PC), and its functional relationship with other signaling genes (especially with p53) in regulating the biological behavior of PC cells. IHC, IF, IB, and real-time PCR were used to detect CRT expression in PC, while transfection and drug intervention were used to investigate the functional relationship of CRT with other signaling genes. IHC showed both CRT and p53 expression was significantly increased in PC, compared to that in paired non-cancerous pancreatic tissues (P < 0.001). High expression of CRT was positively associated with tumor UICC stage and lymph nodes metastasis (P = 0.034 and P = 0.015), and was an independent adverse prognostic indicator in patients with PC. No relationship was found between CRT and p53 expression in spearman's rank correlation test. Altered expression of CRT did not change p53, MDM2, pho-AKT, pho-p38, and pho-JNK expression, but had a specific regulation on pho-ERK. Meanwhile, CRT-regulated cell proliferation, migration, and invasion of PC cells in MEK/ERK pathway dependent manner. In addition, CRT knockdown significantly decreased pho-ERK expression and cell chemoresistance independent of activated p53 and caspase-3-related apoptosis in gemcitabine- or oxaliplatin-treated Capan-2 cells. Our study first demonstrated that overexpression of CRT contributed to the development and progression of PC through MEK/ERK-signaling pathway but independent of p53. The interaction between CRT and MEK/ERK pathway might provide a new idea for revealing malignant biology and supplying new gene targeted chemotherapy of PC.
Our earlier work showed that Musashi (MSI)-2 promoted the development of pancreatic cancer (PC) by down-regulating Numb, which prevented murine double-minute (MDM)-2-mediated p53 ubiquitin degradation. Thus, we investigate the relationship among MSI2, Numb, MDM2, and p53 in PC and, an association that has not been reported to our knowledge. MSI2 had no relationship with mutant p53 (mtp53) and wild-type p53 (wtp53) in normal PC cells. However, in response to gemcitabine or cisplatin treatment, MSI2 silencing simultaneously down-regulated MDM2 and up-regulated Numb and wtp53 protein levels. Moreover, these 4 endogenous proteins can be coimmunoprecipitated as a quaternary complex. Numb small interfering RNA (siRNA) reversed the MSI2 silencing-induced p53 increase. During treatment with chemical agents, MSI2 silencing decreased drug resistance and cell motility and inhibited tumor growth, all of which were significantly reversed by p53 siRNA. MSI2 was also negatively associated with Numb and positively associated with MDM2 expression in tissue. Overexpression of MSI2, MDM2, and mtp53 and weak expression of Numb were closely associated with aggressive clinicopathologic characteristics and poor prognosis for patients with PC. MSI2 negatively regulates wtp53 protein by up-regulating MDM2 and down-regulating Numb after treatment with chemical agents. MSI2 promotes drug resistance and malignant biology of PC in a p53-dependent manner.-Sheng, W., Dong, M., Chen, C., Wang, Z., Li, Y., Wang, K., Li, Y., Zhou, J. Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer.
Musashi2-Numb interaction plays a vital role in the progression of myeloid leukemia. However, its potential role in solid cancers has rarely been reported. We investigated the coordinate function of Musashi2-Numb in the development of pancreatic cancer (PC) in vitro and vivo. Both Musashi2 protein and mRNA levels were higher in PC tissues than that in paired normal pancreas (P<0.05). Musashi2 overexpression and Numb positive expression were positively and negatively associated with tumor size and UICC stage, respectively (P<0.05). Multivariate analysis identified Musashi2 and Numb as adverse and favorable independent indicators for the survival of PC patients. Moreover, patients with high Musashi2 expression combining with negative Numb expression had a significantly worse overall survival (P=0.001). The negative relationship between Musashi2 and Numb was found at both PC tissue and cell levels. These two endogenous proteins can be co-immunoprecipitated from PC cell lines, and Musashi2 silence up-regulated Numb protein in vitro and vivo. Meanwhile, its silence decreased cell invasion and migration in vitro and inhibited the growth of subcutaneous tumors and the frequency of liver metastasis in vivo. However, Numb knockdown significantly reversed the decrease of cell invasion and migration induced by Musashi2 silence. Musashi2 promotes the development and progression of pancreatic cancer by down-regulating Numb protein. The interaction of Musashi2-Numb plays a significant role in the development and progression of PC.
Background Our previous study showed that calreticulin (CRT) promoted EGF-induced epithelial-mesenchymal transition (EMT) in pancreatic cancer (PC) via Integrin/EGFR-ERK/MAPK signaling. We next investigated the novel signal pathway and molecular mechanism involving the oncogenic role of CRT in PC. Methods We investigated the potential role and mechanism of CRT in regulating intracellular free Ca2+ dependent acute and chronic endoplasmic reticulum stress (ERS)-induced EMT in PC in vitro and vivo. Results Thapsigargin (TG) induced acute ERS via increasing intracellular free Ca2+ in PC cells, which was reversed by CRT silencing. Additionally, CRT silencing inhibited TG-induced EMT in vitro by reversing TG-induced changes of the key proteins in EMT signaling (ZO-1, E-cadherin and Slug) and ERK/MAPK signaling (pERK). TG-promoted cell invasion and migration was also rescued by CRT silencing but enhanced by IRE1α silencing (one of the key stressors in unfolded protein response). Meanwhile, CRT was co-immunoprecipitated and co-localized with IRE1α in vitro and its silencing led to the chronic ERS via upregulating IRE1α independent of IRE1-XBP1 axis. Moreover, CRT silencing inhibited IRE1α silencing-promoted EMT, including inhibiting the activation of EMT and ERK/MAPK signaling and the promotion of cell mobility. In vivo, CRT silencing decreased subcutaneous tumor size and distant liver metastasis following with the increase of IRE1α expression. A negative relationship between CRT and IRE1α was also observed in clinical PC samples, which coordinately promoted the advanced clinical stages and poor prognosis of PC patients. Conclusions CRT promotes EMT in PC via mediating intracellular free Ca2+ dependent TG-induced acute ERS and IRE1α-mediated chronic ERS via Slug and ERK/MAPK signaling.
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