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.     

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     

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.     

Paget’s disease of the breast: the experience of the European institute of oncology and review of the literature

Background Paget’s disease of the breast is an uncommon presentation of breast malignancy, accounting for 1–3% of all the breast tumors and presents in different histopathologic patterns: in association with an underlying invasive or non invasive carcinoma, or without any underlying neoplasia. In the literature, different methods are used for the treatment. Mastectomy with or without axillary dissection has been considered as the standard treatment procedure for many years. Several studies have already shown that breast conservation with radiation therapy is an oncologically safe option. Regarding the axillary approach, several studies have documented the presence of positive sentinel lymph node even in Paget’s disease alone. The objective of this study was to retrospectively analyze outcome of patients affected by Paget’s breast disease and to define our institutional experience. Patients and methods Between May 1996 and February 2003, 114 patients with confirmed Paget’s disease of the breast were retrieved and underwent surgery at the European Institute of Oncology of Milan, Italy. The median age of the patients was 54 years at the time of the diagnosis. In our study, the histopathological examination of the operated specimen revealed one hundred seven patients with Paget’s disease associated with an underlying invasive or non invasive carcinoma, and seven patients without underlying carcinoma. Patients underwent either conservative breast surgery or mastectomy, with or without sentinel lymph node biopsy and/or axillary surgery. Each patient was evaluated after surgery at a multidisciplinary meeting to selecting systemic therapy. Results Seven patients had “pure” Paget’s disease of the breast and one hundred seven had the disease associated with an underlying carcinoma. As surgical techniques 71 mastectomies and 43 breast conserving surgeries have been performed. Complete axillary dissection was done in patients with clinically positive lymph node and/or sentinel lymph node biopsy positive. Sentinel lymph node biopsy was performed in nineteen patients with invasive component and five were positive and underwent axillary dissection. Eleven sentinel lymph node biopsies were done in patients with non invasive component and none of them was positive. Adjuvant systemic therapies were based on the final tumor, node and metastasis stage: thirty patients received adjuvant chemotherapy alone, fourteen received endocrine treatment alone, twenty-six patients were evaluated to receive both chemo and endocrine therapy. The median duration of follow up was 73 months and was updated in the last 6 months. Five patients developed local recurrence, one had regional recurrence, another two had loco-regional recurrences and fourteen had distant metastasis as a first event. Malignancy-related deaths were censored in the statistical analyses cancer for and due to another tumor in eleven patients. Additionally, deaths were not related to malignancy totally in thirteen patients. Conclusions Screening examination and imaging techniques are fundamental. Breast conserving surgery combined with breast irradiation for patients with invasive and non invasive breast carcinoma has become the treatment of first choice. All surgical conservative approaches should include the complete nipple–areolar complex and margins of resected specimen free of tumor. Thanks to the evolution of the conservative approach, good cosmetic result can be obtained. To be informed about the axillary lymph node status and to avoid the patient to have a second surgical approach, sentinel lymph node biopsy should be performed.     

Effect of Neoadjuvant CAF Regimen on the Expression of BCSG1 in Breast Cancer

Objective： To evaluate the efficacy of neoadjuvant chemotherapy and explore a sensitive and objective way in the evaluation of neoadjuvant chemotherapy, the pathological changes and BCSG1 expression were studied by pathological and immunohistochemical method in breast cancer patients with CAF neoadjuvant chemotherapy （Cyclophosphamide, Adriamycin and Fluorouracil, CAF） and those without at the same period. Methods： Specimens were obtained from 34 breast cancer patients receiving neoadjuvant CAF regimen chemotherapy （CAF group） and 110 breast cancer patients not receiving neoadjuvant chemotherapy （control group）. The BCSG1 expression was detected by SP immunohistochemistry. Correlation between BCSG1 expression and pathological response to CAF neoadjuvant chemotherapy was analyzed. Results： Overall response rate to neoadjuvant chemotherapy was 79.4%. The strong cytoplasm expression of BCSG1 was significantly lower in CAF group than in control group （29.4% vs. 64.5%, P〈0.01）. In CAF group, the positive cytoplasm expression in partial response （PR） （grade Ⅱ） cases was significantly lower than that in no response （NR） （grade Ⅲ） cases （P=0.002）. Conclusion： Neoadjuvant chemotherapy of CAF regimen could decrease the nuclear expression of BSCG1 in breast cancer.     

Interleukin-1β regulates the migratory potential of MDAMB231 breast cancer cells through the hypoxia-inducible factor-1α

The casual relationship between inflammation and tumour progression has been widely accepted and the etiology of breast cancer has been associated with inflammatory processes. Interleukin (IL)-1β, besides its central role in inflammation, has also been recognised as a powerful player in tumour progression, angiogenesis and invasiveness. Recently, there has been considerable interest in understanding the non-hypoxic upregulation of the hypoxia-inducible factor (HIF)-1α by IL-1 in neoplastic cells since aberrant expression of HIF-1α correlates with tumour progression. Here, using the highly invasive human breast cancer cell line MDAMB231, we studied the effect of IL-1β on tumour cell migration along with HIF-1α accumulation. We observed that non-hypoxic induction of HIF-1α by IL-1β in MDAMB231 was associated with increased cell migration, paralleled by upregulation of p38 MAPK phosphorylation and CXCL8/CXCR1 expression. Inhibition of HIF-1α by siRNA resulted in a significant reduction of CXCR1 expression and IL-1β-induced cell migration in MDAMB231 cells, thus confirming a role of HIF-1α in the non-hypoxic-IL-1β-dependent induction of migratory potentials. Our observation that IL-1 induces HIF-1α accumulation in MDAMB231 cells was confirmed in tumour cells growing in vivo using an experimental approach, mimicking the endogenous release of IL-1 in mice bearing MDAMB231 xenografts. Our in vivo data, along with the fact that inhibition of HIF-1α resulted in the decrease of IL-1β-promoted cell migration, further support the link between inflammation and cancer. The overall results may have important implications in those therapeutic approaches aimed to inhibit IL-1-mediated activities in tumour cells, specifically in breast cancer.     

Potentiation of the antitumor activity of 5-trifluoromethyl-2′-deoxyuridine by the use of depot forms of the parent compound

Summary 5-Trifluoromethyl-2-deoxyuridine (CF₃dUrd), an antitumor agent, is known to be short-lived in human plasma. Since its rapid elimination from the blood-stream seems to have descouraged the clinical evaluation of this drug, we explored the potential use of masked derivatives of CF₃dUrd as depot forms of the parent compound. First, we observed that the toxicity of CF₃dUrd against HeLA cells in culture was 10⁴ times greater for a 24-h treatment as compared with a 1-h treatment at identical concentrations of the drug, which suggests the importance of using a prolonged treatment period. In fact, the divided dosing of CF₃dUrd to L1210-bearing mice was markedly more effective than its single administration. 5-O-Hexanoyl-,N ³-p-butylbenzoyl-, 5-O-benzyloxymethyl-, and 3-O-benzyl-CF₃dUrd were found to be effective in maitaining the CF₃dUrd concentration in plasma. The oral doses of these agents required to achieve 50% growth inhibition (ED₅₀) in mice bearing sarcoma 180 tumors were 19, 34, 10, and 13 mg kg^–1 day^–1, respectively, whereas that of CF₃dUrd was 63 mg kg^–1 day^–1. The ED₅₀ values for these compounds were inversely correlated with the residence time of CF₃dUrd in plasma. The therapeutic indices of these compounds, calculated as the dose producing a 50% inhibition of body-weight gain (IB₅₀) divided by the ED₅₀ value (1.89, 1,21, 1.40, and 2.15, respectively), were significantly higher than that of CF₃dUrd (0.78). Consequently, these depot forms of CF₃dUrd, particularly 3-O-benzyl-CF₃dUrd, are expected to be more useful than the parent compound as antitumor agents.Abbreviations CF₃dUrd 5-trifluoromethyl-2-deoxyuridine - CF₃dUMP 5-trifluoromethyl-2-deoxyuridine-5-monophospate - S180 sarcoma 180 - L1210 L1210 leukemia - k_el elimination rate constant - T1/2 half-life time - AUC area under the curve - ILS increase in life span - TS thymidylade synthase - FdUMP 5-fluoro-2-deoxyuridine-5 monophosphate - FUra 5-fluorouracil     

Are the Phenotypes of Preneoplastic Lesions of Significance for Cancer Prevention? 1. Liver

Preneoplastic lesions have been described for most major sites of human tumour development. They appear to be sharecharacteristics like monoclonality, induction by all classes of carcinogens and some quantitative relationship to actualtumours. Extensive studies of preneoplasia in the liver of the rat has indicated that a directed shift in phenotype occurs,commensurate with greater physiological emphasis on growth potential. One characteristic change is increase in the keyenzyme of the pentose phosphate pathway, glucose 6 phosphatase dehydrogenase as well as elevation in glycolysis, reductionof gluconeogenesis. In general, the changes observed in preneoplastic liver lesions appear reminiscent of the effects ofinsulin or other hormones on hepatocytes, pointing to possible application of specific inhibitors for cancer chemoprevention.     

Association of α1 acidic glycoprotein and squamous cell carcinoma of the head and neck

Serum from patients with different malignancies contain an abnormal concentration of a a1-acidic-glycoprotein (AAG) and also, increased levels of AAG are associated with the presence of tumor mass. In the present report, serum levels of AAG were measured by radial immunodiffusion in squamous cell carcinoma of the head and neck (SCCHN) patients taking into account disease status parameters such as tumor localization, stage and extension of disease. Immunohistochemical methods, SDS-PAGE and Western-blotting were employed to study the expression of AAG and a carbohydrate related antigen (sialyl Lewis x) in tumor tissues and derived fractions. AAG showed abnormal levels in 7/15 oral cavity tumor patients sera, 2/5 oropharynx and 5/10 larynx tumors; increased AAG serum levels belonged to patients with disseminated disease. On the other hand, the presence of AAG and sialyl Lewis x were demonstrated in carcinoma cells and in derived fractions from tumor tissues belonging to patients with elevated AAG serum levels. In the present study, we have found elevated levels of AAG in serum samples from SCCHN patients; these neoplastic cells are capable to express AAG.     

Combined treatment of IL-1α and TNF-α potentiates the antitumour effect of hyperthermia

Vasoactive cytokines, such as IL-1α and TNF-α, modulate the homeostatic state at the endothelial surface and cause various types of pathological damage in vascular systems. We investigated the potential therapeutic effects of IL-1α and TNF-α in combination with hyperthermia on SCK tumours grown in the legs of A/J mice. We first determined the effect of cytokines on tumour blood perfusion with the ⁸⁶Rb uptake method. When the host mice were given an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the tumour blood perfusion markedly declined to 46 and 82% of control, respectively. The combination of IL-1α and TNF-α reduced the ⁸⁶Rb uptake to 41% of control. Hyperthermia at 42·5°C for 1 h reduced the tumour blood flow to 71% of control. The tumour blood perfusion decreased further to 20% of control when the tumours were heated for 1 h at 42.5°C starting 4h after the injection of both IL-1α and TNF-α. The changes in clonogenic cell numbers in SCK tumours, as determined by the in vivo-in vitro assay, following various treatments was also investigated. At 4h after an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the clonogenicity of SCK tumours significantly decreased to 29 or 37% of control, respectively. Heating at 42.5°C for 1h caused a decline in the clonogenic cell number to 30% of control. When both IL-1α and TNF-α were given and tumours were heated 4h later at 42·5°C for 1h, the clonogenic cell number markedly declined to 0.4% of control. The time needed for control tumours to reach 4x their initial volume was about 3 days, and treatment with IL-1α or hyperthermia alone induced a tumour delay growth by about 1 day. The combined injection of IL-1α and TNF-α followed by a heating at 42·5°C for 1h delayed the tumour growth by 6 days. The results in this study suggest that prior impairment of blood circulation by the combined treatment of IL-1α and TNF-α potentiates hyperthermic damage in tumours.     

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer

Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon 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.