Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.     

Inhibition of the Rho/ROCK pathway enhances the efficacy of cisplatin through the blockage of hypoxia-inducible factor-1α in human ovarian cancer cells

Rho, a Ras-related small GTPase, and Rho-associated coiled coil-containing protein kinase (Rho kinase, ROCK1 and ROCK2) are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. Rho/ROCK kinases also play roles in proliferation, differentiation, apoptosis and oncogenic transformation. In the present study, we have shown that Rho/ROCK pathway inhibition by fasudil, an orally administered inhibitor of Rho kinases, enhanced cisplatin-induced growth inhibition and apoptosis in human ovarian cancer cell lines. Fasudil inhibited hypoxia inducible factor (HIF)-1α protein expression. Knockdown of RhoA, ROCK1 or ROCK2 also attenuated the expression of HIF-1α. Furthermore, knockdown of HIF-1α using small interfering RNA enhanced cisplatin-induced growth inhibition and apoptosis as did inhibition of the Rho/ROCK pathway by fasudil, the Rho/ROCK inhibitor Y27632, or by Rho/ROCK knockdown. Therefore, the Rho/ROCK pathway may modulate HIF-1α signal transduction and blockade of Rho/ROCK enhances the efficacy of cisplatin by inhibiting HIF-1α in ovarian cancer cells. Our findings suggested that the Rho/ROCK pathway may be a new target for molecular targeting therapies against ovarian cancer.     

Dissection of the RET/β-catenin interaction in the TPC1 thyroid cancer cell line

The RET receptor tyrosine kinase is a member of the cadherin superfamily and plays a pivotal role in cell survival, differentiation and proliferation. Currently, 12 ret/ptc chimeric oncogenes, characterized by the fusion between the intracellular domain of RET and different activating genes, which can cause ligand-independent dimerization and constitutive activation, have been described. β-catenin is usually involved in the maintenance of cell-to-cell adhesion and mediates the Wnt/β-catenin pathway important during embryogenesis and in cellular malignant transformation. Recently, a novel mechanism of RET-mediated function through the β-catenin pathway has been reported in multiple endocrine neoplasia type 2 and in sporadic thyroid carcinomas. Here, we investigated the effects of the ZD6474, a small molecule RET-inhibitor, on RET/β-catenin interaction. We confirmed the ZD6474 mediated-inhibition of recombinant RET kinase and of growth of cells expressing RET/PTC. Interestingly, we firstly observed reduced cellular mobility and changed morphology of TPC1 treated cells suggesting that RET-inhibitor could affect β-catenin cellular distribution as resulted in its co-immunoprecipitation with E-cadherin. We further investigated this hypothesis showing that TPC1 treated cells displayed predominantly β-catenin cytosolic localization. Surprisingly, RET and β-catenin co-immunoprecipitated in both ZD6474-treated and untreated TPC1 cells, suggesting that RET/β-catenin interaction might not be affected by RET kinase inactivation. All together these results suggest that RET kinase activation is crucial for β-catenin stabilization (pY654), localization and its signaling pathway activation but not for β-catenin/RET physical interactions, in human papillary thyroid carcinomas. In conclusion, ZD6474, by inhibiting RET kinase, down-modulates β-catenin pathway leading its recruitment to the membrane by E-cadherin.     

Small molecule antagonists of the TGF-β1/TGF-β receptor binding interaction

Excessive and inappropriate action of transforming growth factor (TGF)-β has been implicated in the pathogenesis of several disease processes, especially cancer and fibrosis. To identify antagonists of the TGF-β ligand-binding domain that may have therapeutic potential, we screened the National Cancer Institute open access chemical repository for molecules that inhibited binding of TGF-β to the type II receptor (TβRII). About 30,000 molecules were screened resulting in the identification of five structurally related molecules that reduced binding of TGF-β1 to soluble TβRII with an ED50 of approx 10 μM. The chemicals blocked inhibition of Mv1Lu cell growth by TGF-β, TGF-β-induced expression of luciferase driven by the TGF-β response element, and induction of plasminogen inhibitor mRNA detected by Northern blot. In contrast, the chemicals did not block activin-induced inhibition of cell growth. Our results identify a novel chemical group that blocks binding of TGF-β to its receptor and may result in novel treatment for disease. J.K.B. has submitted a patent application for the use of these molecules for blocking TGF-β.     

Beta androstenediol mitigates the damage of 1 GeV/n Fe ion particle radiation to the hematopoietic system

Space exploration is associated with exposure to 1-3 Gy solar particle radiation and galactic cosmic radiation that could increase cancer rates. Effective nontoxic countermeasures to high linear energy transfer (LET) radiation exposure are highly desirable but currently not available. The aim was to determine whether a single subcutaneous injection of androstenediol (Δ(5) androsten-3β, 17β-diol [AED]) could mitigate and restore the mouse hematopoetic system from the radiation-mediated injury of 3 Gy whole-body high LET (56)Fe(26+) exposure. The findings show that postradiation AED treatment has an overall positive and significant beneficial effect to restore the levels of hematopoeitic elements (p<0.001). Androstenediol treatment significantly increased monocyte levels at days 4, 7, and 14 and, similarly, increased red blood cell, hemoglobin, and platelet counts. Flow cytometry analysis 14 days after radiation and AED treatment demonstrated an increase (p<0.05) in bone marrow cells counts. Ex vivo osteoclastogenesis studies show that AED treatment is necessary and advantageous for the development and restoration of osteoclastogenesis after radiation exposure. These findings clearly show that androstenediol functions as a countermeasure to remedy hematopoeitic injury mediated by high LET iron ion radiation. Presently, no other agent has been shown to have such properties.     

Can inhibition of the SDF-1/CXCR4 axis eradicate acute leukemia?

Poor prognosis of acute leukemia with current treatments is mainly due to the relapse of the disease following chemotherapy. In the last decade, an emerging concept has proposed that the leukemia stem cells (LSCs) and their interactions with the BM microenvironment are the major cause of the acute leukemia relapse. Adhesion to the stromal niche is crucial for LSCs as it directly supports self-renewal, proliferation, arrest of differentiation and protects from damaging chemo-agents. One of the key players in this crosstalk between leukemic cells and the BM stroma niche is the chemokine SDF-1. SDF-1 regulates the process of homing and engraftment of LSCs into the BM and inhibition of its receptor CXCR4 induces leukemic cell mobilization into the circulation. However, besides its chemotactic and adhesive functions, SDF-1 is also a pleiotropic cytokine that regulates leukemic cell proliferation as well as their program of differentiation. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies, which demonstrate that blocking CXCR4 is a novel promising approach of therapy. In this review, we focus on the multifaceted SDF-1/CXCR4 axis in acute leukemia and discuss how targeting this pathway could provide potential interest to eradicate the LSCs.     

Deletion of exons 1–3 of the MEN1 gene in a large Italian family causes the loss of menin expression

Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant disease, characterized by parathyroid adenomas, endocrine gastroenteropancreatic tumors and pituitary adenomas, due to inactivating mutations of the MEN1 gene (chromosome 11q13). MEN1 mutations are mainly represented by nonsense, deletions/insertions, splice site or missense mutations that can be detected by direct sequencing of genomic DNA. However, MEN1 patients with large heterozygous deletions may escape classical genetic screening and may be misidentified as phenocopies, thereby hindering proper clinical surveillance. We employed a real-time polymerase chain reaction application, the TaqMan copy number variation assay, to evaluate a family in which we failed to identify an MEN1 mutation by direct sequencing, despite a clear clinical diagnosis of MEN1 syndrome. Using the TaqMan copy number variation assay we identified a large deletion of the MEN1 gene involving exons 1 and 2, in three affected family members, but not in the other nine family members that were to date clinically unaffected. The same genetic alteration was not found in a group of ten unaffected subjects, without family history of endocrine tumors. The MEN1 deletion was further confirmed by multiplex ligation-dependent probe amplification, which showed the deletion extended from exon 1 to exon 3. This new approach allowed us to correctly genetically diagnose three clinical MEN1 patients that were previously considered as MEN1 phenocopies. More importantly, we excluded the presence of genetic alterations in the unaffected family members. These results underline the importance of using a variety of available biotechnology approaches when pursuing a genetic diagnosis in a clinically suggestive setting of inherited endocrine cancer.     

Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-β-d-arabinofuranosyluracil

Summary Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200–1000 m) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 m ara-C following the exposure of cells to ara-U (1mm) resulted in 4.5 pmol ara-C DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 m ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10–100 m) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.Abbreviations ara-C 1--d-arabinofuranosylcytosine - ara-U 1--d-arabinofuranosyluracil - ara-CTP 1--d-arabinofuranosylcytidine triphosphate - HiDAC high-dose ara-C - HiCAU high concentrations of ara-U - dCTP deoxycytidine triphosphate - HiDAU high-dose ara-U - FiTC Fluoroisothiocyanate - dUDP deoxyuridine diphosphate - dUTP deoxyuridine triphosphate - dTTP thymidine triphosphate - BrdUrd bromodeoxyuridine - dCyd kinase deoxycytidine kinase Supported in part by grant CH-35H from the American Cancer Society, by Public Health Service grant CA-12197 from the National Cancer Institute, National Institutes of Health, and by the Gaston Cancer Society     

Anti–PD-1/PD-L1 antibodies in non-small cell lung cancer: the era of immunotherapy

Introduction: Advanced non-small cell lung cancer (NSCLC) has been conventionally treated with cytotoxic chemotherapy with short-lived responses and significant toxicities. Monoclonal antibodies to programmed death-1 receptor (PD-1) and programmed death ligand 1 (PD-L1) have shown tremendous promise in the treatment of advanced NSCLC in various clinical trials.

Areas covered: In this article, we will review the outcomes of various trials of anti-PD-1/anti-PD-L1 antibodies in the treatment of NSCLC. We will also discuss their mechanism of action and toxicities.

Expert commentary: Anti-PD-1/PD-L1 antibodies offer several advantages including significant antitumor activity, induction of long lasting responses, and favorable safety profile. Several trials are now being conducted to evaluate their efficacy as first line agents as well as in combination with other agents. More research is also needed to identify other biomarkers, in addition to PD-L1 expression, that could more reliably predict response to these drugs, and aid in better patient selection.     

Is adenocarcinoma/large cell carcinoma the most radiocurable type of cancer of the lung?

Radiation therapy is widely considered the primary treatment for inoperable "non-small" cell carcinoma of the lung. In clinical investigations, distinction has been infrequent among the histopathologic subtypes of non-small cell carcinoma. Studies have shown significant differences between squamous cell carcinoma and adenocarcinoma/large cell carcinoma; adenocarcinoma/large cell carcinoma has a greater propensity for extrathoracic dissemination, especially to the brain, and it is less curable by resection when regional lymph node metastases are present. No differences have been documented between adenocarcinoma and large cell carcinoma. A retrospective study was undertaken to determine the results of definitive radiation therapy by histopathologic subtype of non-small cell carcinoma of the lung. Between July 1977 and April 1983, 134 patients with non-small cell carcinoma of the lung underwent definitive radiation therapy with curative intent. All patients had performance status scores of 80 to 100 (Karnofsky), and received minimum total doses within the tumor of 60 Gy in 6 to 7 weeks, five fractions per week. The median period of observation was 63 months. Ninety patients had squamous cell carcinoma; 44 had adenocarcinoma/large cell carcinoma. The two groups of patients were comparable in respect to age and Stage; there were significantly more women with adenocarcinoma/large cell carcinoma (27%) than with squamous cell carcinoma (13%). The median survival for patients with squamous cell carcinoma was 11.5 months; the 2 and 4 year survival rates were 21 and 7%, respectively. The median survival for patients with adenocarcinoma/large cell carcinoma was 18 months; 2 and 4 year survival rates were 38 and 23%, respectively. Comparison of the overall survival experience did not show a significant difference between the two cell types (p = .12 using Gehan's generalized Wilcoxon test). However, comparison of the proportion of patients with adenocarcinoma/large cell carcinoma surviving 18 months (50%) was significantly higher (p = .02) than that with squamous cell carcinoma (30%). A small body of data from the literature also suggests a better long-term prognosis for adenocarcinoma/large cell carcinoma. This observation requires confirmation from large trials with histopathologic review. If it is confirmed, there are important implications for therapeutic strategies in future clinical investigations of inoperable carcinoma of the lung.     

Metformin inhibits gastric cancer via the inhibition of HIF1α/PKM2 signaling

Recent evidence suggests that anti-diabetic drug metformin prevents cancer progression, but the mechanism by which metformin inhibits tumor growth remains elusive. In this study, we investigated the anticancer role of metformin in gastric cancer and explored the underlying mechanism. The expression of hypoxia inducible factor 1α (HIF1α) and pyruvate kinase M2 (PKM2) in different stages of gastric cancer tissues was detected by immunohistochemistry. Gastric cancer cell viability was evaluated by CCK-8 assay; apoptosis and cell cycle were analyzed by flow cytometry. The expression of PI3K, Akt, HIF1α, PARP, PKM2 and COX in gastric cancer cells was detected by immunofluorescence and Western blot analysis. We found that HIF1α and PKM2 protein expression levels were higher in advanced gastric cancer tissues than in gastritis tissues. Metformin reduced gastric cancer cell viability, invasion and migration. Metformin induced apoptosis and cell cycle arrest in part through inhibiting PARP expression. Metformin downregulated PI3K, Akt, HIF1α, PARP, PKM2 and COX expression. Moreover, overexpression of HIF1α increased gastric cancer cell viability, invasion and migration. In summary, metformin has profound antitumor effect for gastric cancer by inducing intrinsic apoptosis via the inhibition of HIF1α/PKM2 signaling pathway.     

Discussion of the 8th edition of AJCC/UICC staging system from the clinical stage Ⅲ nasopharyngeal carcinoma

Objective To evaluate the 8^th edition of AJCC/UICC staging system for stage Ⅲ nasopharyngeal carcinoma (NPC) by the survival analysis. All patients were treated with intensity-modulated radiotherapy (IMRT). Methods Among 1351 treatment-naïve NPC patients who received radiotherapy/chemoradiotherapy in our hospital from December 2008 to October 2014, 742 and 784 cases were classified as clinical stage Ⅲ based on the criteria of the 7^th and 8^th edition of AJCC/UICC staging systems, respectively. These patients were classified into three subgroups according to the 7^th and 8^th edition of AJCC/UICC staging systems:T3N0-1 as G1(n=226, n=245), T1-2N2 as G2(n=180, n=187) and T3N2 as G3(n=336, n=352). The 5-year overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS) and local-regional recurrence-free survival (LRRFS) were analyzed with Kaplan-Meier method. The differences among different groups were evaluated by log-rank test. Results There were 93.6% patients evaluated by the 8^th AJCC/UICC staging system remained the same cohort with those by the 7^th AJCC/UICC staging system. The 5-year OS, PFS, DMFS and LRRFS of the 8^th and 7^th staging systems were 84.8% and 85.4%, 76.2% and 77.0%, 80.4% and 81.3%, 89.8% and 90.6%, respectively (all P>0.05). The OS, PFS or DMFS significantly differed among three subgroups classified by the 8^th staging system (all P<0.001). In addition, statistical significance was observed between G1 and G2, and between G1 and G3(both P<0.05), whereas no statistical significance was noted between G2 and G3(P=0.183, 0.310, 0.248). Conclusions The distribution features and clinical endpoints of clinical stage Ⅲ defined by the 8^th AJCC/UICC staging system are similar to those defined by the 7^th AJCC/UICC staging system. The distribution of survival risk significantly differs among different subgroups. N2 plays a major role in assessing the survival risk of patients with stage Ⅲ NPC. In the era of IMRT plus chemotherapy, the effect of local tumors on clinical prognosis has been diminished. The 8^th AJCC/UICC staging system remains to be further improved.     

Two novel variants in the 3′UTR of the BRCA1 gene in familial breast and/or ovarian cancer

For the majority of breast and/or ovarian cancer patients tested for BRCA1/2 genes, mutation screening of the coding regions remains negative. MicroRNAs which negatively regulate mRNA translation by binding to 3′ untranslated region (3′UTR) are implicated in cancer. Genetic changes in the 3′UTR of several genes were reported to be associated with higher susceptibility to particular tumor types. The aim of this study was to analyze the BRCA1 3′UTR in patients tested negative for BRCA1/2 deleterious mutations, in order to find variants implicated in the decrease of BRCA1 expression through modification of miRNA binding. Genotyping analyses were performed on genomic DNA of 70 BRCA negatives index cases, selected among patients with breast or ovarian cancer, less than 50 years old, with a strong family history. The co-occurrence of the identified variants with deleterious BRCA1 mutations was then determined in a control population of 210 patients. A luciferase gene reporter assay was used to investigate the impact of the variants on the BRCA1 gene expression. Two novel variants, c.*750A>G and c.*1286C>A, were identified in the 3′UTR of BRCA1 gene, in two patients. The former was found three times in the control population, whereas the latter was absent. The used functional assay did not reveal any effect on the luciferase expression. This study reveals a weak genomic variability in the 3′UTR of the BRCA1 gene. All together, the results led us to classify the variant c.*750A>G as probably neutral, the variant c.*1286C>A remaining unclassified.     

Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway

Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the Apc^Min/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/β-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.     

Enhancement of in vitro cell motility and invasiveness of human malignant pleural mesothelioma cells through the HIF-1α-MUC1 pathway

In this study, we examined the effects of hypoxia on the malignancy of human malignant pleural mesothelioma (MPM) cell lines, and found (1) hypoxia enhanced motility and invasiveness of human malignant pleural mesothelioma (MPM) cells; (2) this phenomenon resulted from increased expression of sialylated MUC1 through the activation of HIF-1 pathway; (3) two HIF-binding sites located in the promoter region of MUC1 were important for MUC1 transactivation under hypoxia. These findings are useful for better understanding molecular mechanisms of aggressive behavior of MPM cells and for targeting them in the clinical therapies for MPM patients.     

Impact of Clinicopathologic Features on the Efficacy of PD-1/PD-L1 Inhibitors in Patients With Previously Treated Non–small-cell Lung Cancer

Background

The current study aimed to comprehensively investigate the impact of various clinicopathologic features on the efficacy of programmed cell death-1 (PD-1) and ligand (PD-L1) inhibitors in patients with previously treated non–small-cell lung cancer (NSCLC).